Re: [ccp4bb] AW: Strange cysteines

2021-12-22 Thread Roger Rowlett
Another possibility to model is a couple of S-hydroxy cysteines. We've seen that in a Cys-amidase where there was artifactual oxidation damage to the protein during storage or RT crystallization. Roger Rowlett On Wed, Dec 22, 2021, 8:00 AM Andrew Purkiss wrote: > As you say, the Cys are

Re: [ccp4bb] off-topic: pH meters

2021-09-20 Thread Roger Rowlett
they were much more difficult for my students to break, and they last for years. (My students never succeeded in breaking one.) Gel-filled electrodes are garbage. Refillable double junction electrodes are the way to go for stable readings, especially in Tris, and minimizing heavy metal contamination. Roger

Re: [ccp4bb] Enzyme Vmax and Km

2021-06-18 Thread Roger Rowlett
of rate vs. substrate data. For true product inhibition, there are several possible models that could be considered, based on the behavior of the data. Consultation with an enzyme kineticist might be warranted for for complex behavior. Roger Rowlett On Fri, Jun 18, 2021, 3:53 AM Harmer, Nicholas wrot

Re: [ccp4bb] Looking for proteins for undergraduate biochemistry lab

2021-06-16 Thread Roger Rowlett
Human carbonic anhdydrase II is very expressible in *E. coli,* and purifiable in one step via affinity chromatography with para-aminobenzenesulfonamide affinity resin (which is relatively easy to make, and reusable for many years.) It can be assayed by stopped-flow spectrophotometry for CO2

Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

2020-12-09 Thread Roger Rowlett
Salt concentrations less than 100 mM can lead to nonspecific adsorption to the gel exclusion media, potentially leading to band broadening, and delayed elution. Overloading gel exclusion columns (more than 2-4% Vt) can also lead to elution band artifacts. Check these issues first. Roger Rowlett

Re: [ccp4bb] electron density close Histidine side chain

2020-07-20 Thread Roger Rowlett
Almost certainly a metal ion, possibly Ni(2+) if a Ni-NTA column was used for purification. Ni-N bond lengths are typically around 2.0 A. Additional density is probably coordinated water molecules, which should have similar Ni-O bond distances around 1.9 A. It is fairly common to find adventitious

Re: [ccp4bb] insertion of a chemical

2020-06-30 Thread Roger Rowlett
, Roger Rowlett On Tue, Jun 30, 2020, 7:25 AM Daniele Veggi wrote: > Hi Yadav, > yes, this is only a representation of what I would like to do. If during > the refinement will continue to appear density, probably I'm on the goo

Re: [ccp4bb] insertion of a chemical

2020-06-30 Thread Roger Rowlett
suggestive of a thioketal adduct or a simple disulfide bridge. Building the thioketal, if actually present, will require addition skill with ligand building and Coot. You are essentially attaching an isopropyl group to the two Cys sulfur atoms. Roger Rowlett Gordon & Dorothy Kline Professor,

Re: [ccp4bb] Molecular replacement problem

2020-06-18 Thread Roger Rowlett
was reasonable and space group was likely correct, and that I had the correct number of chains per ASU. My initial MR attempts, based on Matthew's number estimates, were 2 chains short, which was obvious from crystal packing. Cheers, and good luck! Roger Rowlett Dorothy & Gordon Kline Professor, Emer

Re: [ccp4bb] Covalent Cysteine Aduct

2020-03-27 Thread Roger Rowlett
S-hydroxycysteine can cartainly show up under nonreducing storage or crystallization conditions, as an artifact. Cheers, Roger Rowlett On Fri, Mar 27, 2020, 5:45 PM Chris Fage wrote: > Hi Richard, > > I recently observed the sulfenic acid derivative of a cysteine residue > (S

Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Wincoot middle-mouse button does not work

2020-03-23 Thread Roger Rowlett
-click. Again, you have to go into the mouse configuration settings. I have two mice (a 3-button and a 2-button) configured to work properly with Wincoot in Win10. Roger Rowlett Gordon & Dorothy Kline Professor, Emeritus Colgate University On Mon, Mar 23, 2020, 11:56 AM Schreuder, Herman

Re: [ccp4bb] A question of density

2020-03-04 Thread Roger Rowlett
Chloride coordination spheres are typically tetrahedral (CN=4) or square pyramidal (CN=5), and occasionally octahedral (CN=6). Arg and His and well as amide nitrogens are common protein ligands, and it is possible but not common to see carboxylates in the coordination sphere. Roger Rowlett

Re: [ccp4bb] A question of density

2020-03-04 Thread Roger Rowlett
? Roger Rowlett Gordon & Dorothy Kline Professor, Emeritus Colgate University On Wed, Mar 4, 2020, 12:20 PM Nukri Sanishvili wrote: > Hi Jessica, > You do not say how well is the rest of the structure refined. > First, you should refine the structure best you can, without placi

Re: [ccp4bb] Protein concentration for the initial crystallisation trials

2020-01-08 Thread Roger Rowlett
. by manipulating protein/screen volume ratio. __ Roger Rowlett On Wed, Jan 8, 2020, 11:16 AM Armando Albert wrote: > Dear all, > I was wondering how to guess the optimal protein concentration for the > initial crystallisation trials. Is there any trick or assay o

Re: [ccp4bb] difficult molecular replacement solution

2019-11-13 Thread Roger Rowlett
to simplify the search. Good luck. __ Roger Rowlett On Wed, Nov 13, 2019, 10:33 AM Robert S Phillips wrote: > I have been working on a protein structure which has been hard to solve by > molecular replacement. > > Unit cell: (60.6, 172.34, 196.42, 90, 90, 90) > Sp

Re: [ccp4bb] crysalis pro from rigaku

2019-11-04 Thread Roger Rowlett
The current version of CrysalisPro will directly import several image formats, or you can convert images to Esperanto format to import. The latter is a bit clumsy but does work. __ Roger Rowlett On Mon, Nov 4, 2019, 9:54 AM Almudena Ponce Salvatierra <>

Re: [ccp4bb] ITC question -dimer vs monomer

2019-10-03 Thread Roger Rowlett
Won't this depend on the relative final concentrations of A and B in the two experiments? If A going into excess B will have different mass action considerations that B going into excess A. Even if the final concentrations of A and B are stoichiometric, the initial stages of the titration will

Re: [ccp4bb] Another difficult MR case

2019-08-29 Thread Roger Rowlett
numbers, though, so 2 is not a great improvement, e.g. __ Roger Rowlett On Thu, Aug 29, 2019, 1:42 PM Jonathan Cooper <> wrote: > It would be useful to know the number of molecules per asymmetric unit and > the sequenc

Re: [ccp4bb] low resolution map with unmodelled map

2019-08-19 Thread Roger Rowlett
, then there are ways of using noncrystallographic symmetry in your ASU to significantly improve your initial maps for rebuilding, although sequence assignment may still be challenging at this resolution. Roger Rowlett On Mon, Aug 19, 2019, 8:15 PM Zhu Qiao wrote: > Sorry for the initial message. I tried to att

[ccp4bb] FFT bug, CCP4i job file configuration

2019-08-06 Thread Roger Rowlett
FYI, When FFT is run in the CCP4i interface, the output file destination is configured incorrectly. Examining the batch file that is created for the job, it appears that CCP4i ignores the MAPOUT filename and substitutes the file name of the temporary file, which is (alas) deleted in the last

[ccp4bb] Position opening - Colgate University

2019-08-03 Thread Roger Rowlett
Please find attached a description of a position opening at Colgate University that may be of interest to a protein crystallographer interested in working with undergraduates in a research-rich, research supportive environment. Colgate is equipped with an Oxford Diffraction home source, Gryphon

Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

2019-07-19 Thread Roger Rowlett
If it is as it appears, it is disappointing to see this in JACS. I would expect better. Unfortunately, reviewers don't always get a lot of information to judge quality of structures (which has been discussed extensively  on prior occasions on this board), so some trust is involved that what

Re: [ccp4bb] analytical gel filtration columns

2019-04-17 Thread Roger Rowlett
How about BioGel-A50m with an exclusion limit of 50 million Da? I've used A15m for complexes as large as 500 kDa and it seemed to work well. __ Roger Rowlett On Wed, Apr 17, 2019, 11:11 AM Mohinder Pal wrote: > Dear all, > > I would like to gel filter a multi protei

Re: [ccp4bb] SO4 or PO4

2019-02-16 Thread Roger Rowlett
donor to acceptor hydrogen bonding contacts. Sulfate rarely has donor to acceptor hydrogen bonding contacts, as it is SO4= at any reasonable pH. Roger Rowlett On Sat, Feb 16, 2019, 4:06 AM 张士军 < wrote: > Dear all > > I have got a crystal grown at the co

Re: [ccp4bb] Sulphate or phosphate?

2018-07-31 Thread Roger Rowlett
You might be able to distinguish sulfate from phosphate by examining hydrogen bonding partners. Phosphate can donate one or two hydrogen bonds at neutral pH values, whereas sulfate is usually only a hydrogen bond acceptor. (Having said that, we have published a structure where a sulfate clearly

Re: [ccp4bb] Sulphur SAD at home source

2018-04-03 Thread Roger Rowlett
Iodine is ideally suited for Cu K-alpha SAD phasing, and iodide ions can normally be easily added by soaking crystals in potassium iodide containing solutions, which can be done at the time of cryopreservation. A quick lit search will turn up the appropriate protocols. For structural genomics

Re: [ccp4bb] Yet another "what's my blob" thread

2017-10-02 Thread Roger Rowlett
-. Blob 2 is less likely to be metal around those Arg residues, but Cl- or bicarbonate are possible and well-known in this kind of coordination environment. Cl- is in your mixture, and bicarbonate can accumulate, especially at alkaline pH from atmospheric CO2. Cheers, Roger Rowlett On Oct 2

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

2017-07-12 Thread Roger Rowlett
Besides empirically adjusting the weighting factor for X-ray data to increase the geometric constraints, have you tried jelly-body refinement or refinement with external constraints? The latter two methods can be helpful when refining low resolution data. Roger Rowlett On Jul 12, 2017 7:33 PM

Re: [ccp4bb] crystallization optimization

2017-07-12 Thread Roger Rowlett
. Cheers, Roger Rowlett On Jul 12, 2017 12:01 PM, "Alun R Coker" <> wrote: > So, if we have a commercial 96 well screen where more than around 60% of > the drops precipitate. It may be worth diluting the whole screen say (30ul > screen and 20ul water in

Re: [ccp4bb] Dynamic light scattering instrument

2017-05-02 Thread Roger Rowlett
We've had a Malvern Zetasizer for many years. ___ 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:

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Roger Rowlett <>> wrote: A bit off topic, but I’ve always wondered how TRIS got so popular what with it’s pKa of 8.3—does anyone know? JPK *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>] *On Be

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Roger Rowlett
What are you dialyzing against? Your storage solution should typically be buffered away from the pI and contain at least a small amount of kosmotropic salt, e.g. NaCl. Some proteins will require additional stabilizing/solubilizing agents such as glycerol or reducing agents. FYI, Tris-Cl, pH

Re: [ccp4bb] Nitrate versus Carbonate

2016-11-11 Thread Roger Rowlett
Once again, chemical intuition may help. At neutral pH values, sulfate is going to be present at SO4(2-), whereas phosphate will be present as H2PO4(-) or HPO4(2-). The hydrogen bond network supporting binding may be able to offer clues. Sulfate is not likely to have any H-bond acceptors in

Re: [ccp4bb] Nitrate versus Carbonate

2016-11-10 Thread Roger Rowlett
. Bicarbonate ions will populate this site in crystals after a few minutes soak at 100 mM concentration. The same crystals soaked in as much as 1M (!) nitrate overnight do not bind nitrate ion in this site. Roger Rowlett On Nov 10, 2016 3:42 PM, "Keller, Jacob" <>

Re: [ccp4bb] Two SGs in one droplet?

2016-10-28 Thread Roger Rowlett
I've seen this kind of thing before. Case 1: two crystal forms in the same droplet, C2 and C222. If you looked closely, you could tell them apart and I was pretty good at getting a high percentage of the desired space group by looking at the crystal forms. The C2 form diffracted better, so I

Re: [ccp4bb] suggestion for structure solution of a protein with low sequence identity

2016-10-25 Thread Roger Rowlett
I'll also recommend Buccaneer. You might try using a combination of PARROT for density modification and NCS averaging followed by autobuilding with BUCCANEER using initial phases from your MR solution. You only have two copies of the protein in the ASU, so you only get a modest boost in

Re: [ccp4bb] Structural biology software that does not run on Windows or gives important Windows-specific problems

2016-10-14 Thread Roger Rowlett
Except for graphics-intensive programs (e.g., Coot, Pymol), it is possible to run Linux within a VM in windows, and you can even share files with the other OS. I actually do it the other way 'round, running Windows in Linux via wine to have a one-box solution for processing data from our

Re: [ccp4bb] Ambiguous metal ion at active site.

2015-07-09 Thread Roger Rowlett
If the metal center is a stable complex then ICP-OES or XRF (e.g. TXRF) methods can easily identify and quantify metals present in a small sample of protein. Roger Rowlett Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three

Re: [ccp4bb] Phaser solution and solvent content for arp warp

2015-06-29 Thread Roger Rowlett
I think I would be tempted to chainsaw one of the ensemble chains of 2IT8 (they look very similar except for side-chain disposition) and use 1 or 2 of these as search models in a Phaser run. If this works, you should see good Z-values and the final result inspected in Coot should show good

Re: [ccp4bb] distorted phosphate molecule geometry after refinement

2015-06-22 Thread Roger Rowlett
:// Shane On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett wrote: I possibility is a M-His(2)-Lys-(OH2) site. Possible metal ions would include Zn(II), although Lys is a relatively rare ligand in zinc

Re: [ccp4bb] distorted phosphate molecule geometry after refinement

2015-06-22 Thread Roger Rowlett
:// Shane On Mon, Jun 22, 2015 at 2:15 PM, Roger Rowlett wrote: I possibility is a M-His(2)-Lys-(OH2) site. Possible metal ions would include Zn(II), although Lys is a relatively rare ligand in zinc

Re: [ccp4bb] distorted phosphate molecule geometry after refinement

2015-06-22 Thread Roger Rowlett
I possibility is a M-His(2)-Lys-(OH2) site. Possible metal ions would include Zn(II), although Lys is a relatively rare ligand in zinc-metalloenzyme sites. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry

Re: [ccp4bb] CCP4 installation problem

2015-06-16 Thread Roger Rowlett
Appu, You will have to edit your .tcshrc (startup) file to point to the correct setup script with a line like this: source /usr/local/xtal/ccp4-6.5/ccp4.setup-csh If you are using a different shell, e.g. bash, you will have to edit the appropriate startup file, e.g. .bashrc. You will

Re: [ccp4bb] Easy way to generate symmetry-related protein chains?

2015-05-22 Thread Roger Rowlett
If you mean generation of pdb coordinates of specific symmetry chains (not just viewing) then you can do this with the symexp command in pymol. Select the desired symmetry partners and save as pdb. You may want to edit duplicate chain id labels in coot or a text editor. Roger Rowlett On May 22

Re: [ccp4bb] CYS mod

2015-05-13 Thread Roger Rowlett
Are the Cys residues in question on the surface of the protein? DMSO (which is in the crystallization mix) is a weak oxidant and could conceivably contain impurities like dimethylsulfide and methanethiol which could form difulfides with surface Cys residues. Or there could have been sulfides

Re: [ccp4bb] Enzyme kinetics

2015-05-08 Thread Roger Rowlett
In these coupled enzyme assays, concentrations of [E1], [S], [E2] and [NADH] need to be chosen such that the rate of the second reaction is at least one to two orders of magnitude faster than the first, otherwise the measured rate -d[NADH]/dt will not be rate-limited by -d[S]/dt. Normally this

Re: [ccp4bb] Off topic - Mercury Lamp for Akta

2015-05-05 Thread Roger Rowlett
I wouldn't mind knowing how to source this lamp as well. But FYI, the lamps are usable for years after the FPLC gives you the dire low intensity warning. We just ignore it until the lamp goes completely dark or it's impossible to measure normal absorbances. We've used the current lamp for

Re: [ccp4bb] cryo condition

2015-05-04 Thread Roger Rowlett
We rarely use glycerol anymore, because it seems to fail so often for many of our current proteins. Try glucose, 25-30%. This is most conveniently done by weighing 125-150 mg of glucose in a microcentrifuge tube, then addding well solution to the 0.5 mL mark and mixing until completely

Re: [ccp4bb] Cleaved peptide density!

2015-04-22 Thread Roger Rowlett
If your protease depends on an oxyanion hole for stabilizing the transition state, fluoride ions are known to be a potent inhibitor of these proteases. (It is a quasi-diagnostic test for serine-type proteases, and related cysteine proteases.) This might allow you to get reactant bound without

Re: [ccp4bb] HPLC vs FPLC for protein purification

2015-04-20 Thread Roger Rowlett
On Apr 20, 2015 8:28 PM, Roger Rowlett wrote: Depends on what you want to accomplish... If you have a liter of crude lysate, capacity should be one of the choices. A step gradient is fast but low resolution; a gradient elution has more resolution but will eat buffer

Re: [ccp4bb] HPLC vs FPLC for protein purification

2015-04-20 Thread Roger Rowlett
rate and possibly capacity. Higher pump pressures are required to get flow through columns packed with tiny particles sizes. To borrow an analytical chemistry maxim: speed, resolution, capacity: pick any two. Roger Rowlett On Apr 20, 2015 6:45 PM, xaravich ivan wrote: Hi

Re: [ccp4bb] Crystallisation of a minority fraction monomers

2015-04-08 Thread Roger Rowlett
The problem with crystallization is that is selects for the least soluble, most packable species. Sometimes that works against what you would like to know. That could include oligomerization state as well as conformational state. For example, some of the allosteric carbonic anhydrases

Re: [ccp4bb] Number of Molecules in Asymetric Unit

2015-04-01 Thread Roger Rowlett
In the CCP4i GUI you will find Phaser Cell Content Analysis under the Analysis tab. IIRC, this calls up the Matthews Probability Calculator. It will give you a good idea of the likely number of search models in the ASU. For large numbers, e.g. 3, the most probable number is likely to be

Re: [ccp4bb] No MR solution

2015-04-01 Thread Roger Rowlett
sense to search for the domains separately? Did you truncate the model side chains? There are lots of ways to skin the cat depending on the problems encountered. 3.0 A may be challenging for a low identity search model. __ Roger Rowlett Gordon Dorothy Kline Professor Department

Re: [ccp4bb] Cis-peptide bond checking

2015-02-16 Thread Roger Rowlett
Great reminder. But there are real non proline cis peptide bonds, including highly conserved motifs in active sites, e.g. 3UAO and its homologs. I would hope these don't get corrected. Roger Rowlett On Feb 16, 2015 5:09 AM, Tristan Croll wrote: Dear all, My

Re: [ccp4bb] how to reduce protein solubility

2015-02-16 Thread Roger Rowlett
Bringing the pH closer to the measured pI would definitely help. The pI can be measured on an IEF gel. Glycosylation, if applicable, could dramatically increase solubility. Roger Rowlett On Feb 16, 2015 11:33 PM, Mattiroli,Francesca wrote: Hi all, I am

Re: [ccp4bb] adding a Cobalt atom within Coot

2015-02-02 Thread Roger Rowlett
This is a known bug in Coot 0.7.x. You can add the metal ion correctly using the Get Monomer dialog and selecting CO as the monomer. This is fixed in Coot 0.8.1. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University

Re: [ccp4bb] PHASER MR solution

2015-01-28 Thread Roger Rowlett
, 2015 at 5:00 PM, Roger Rowlett wrote: Did you search all 8 possibilities of screw axes, e.g. P2221, P21212, P212121, etc? Roger Rowlett On Jan 25, 2015 4:50 AM, jeorgemarley thomas

Re: [ccp4bb] Queries regarding bead beater and french press.

2015-01-12 Thread Roger Rowlett
The Bead Beater has a 15, 40, and 350 mL chambers. I haven't used mine to homogenize yeast, but I suspect it is similar in performance to E. coli disruption. (Different bead sizes are used for yeast than bacteria.) We get excellent, gentle disruption of E. coli in 8 minutes total. A French

Re: [ccp4bb] Some advices on model modification

2015-01-06 Thread Roger Rowlett
How you approach this will depend substantially on the sequence identity between your target and your potential MR models. Definitely remove all non-protein atoms from your search model, as these are not likely at all to be present, or present at these positions, in your target. In addition to

Re: [ccp4bb] CrystalClear tape rolls

2014-12-01 Thread Roger Rowlett
I use 3 clear duck tape. Amazon has it in packages of 6 rolls. Seems to work OK, with maybe one well in a 2x96 well screen interacting with the adhesive. I think it's an isopropanol or dioxane condition. Roger Rowlett On Dec 1, 2014 5:32 AM, Mark J van Raaij wrote: Dear

Re: [ccp4bb] workstation crystallography

2014-11-11 Thread Roger Rowlett
I'm running home-built Ubuntu boxes with old, plain-vanilla CPUs (e.g., Q9300 or core i3/i5/i7) and 6-8Gbyte of RAM, and a cheap Nvidia video card (e.g. GT 9xxx or GT 620).This is more than sufficient to do routine structure solution. Any contemporary desktop or laptop computer should be

Re: [ccp4bb] Adding water molecule and metal atom

2014-10-24 Thread Roger Rowlett
Adding metal ions will work properly through the Get Monomer dialog in 0.7.2. Until Coot is updated (either manually or through a new CCP4 release) this is a reasonable workaround. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry

Re: [ccp4bb] Adding zink and coper by COOT

2014-10-23 Thread Roger Rowlett
There is a bug in version 0.7.2 Coot that causes metal ions added via the place atom at pointer to be a water. However, if you add the metal ions through the Get Monomer dialog I think it will work OK. ___ Roger S. Rowlett Gordon Dorothy Kline Professor

Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!

2014-10-17 Thread Roger Rowlett
You've tried the obvious things. Lowering the IPTG concentration doesn't really work all that well, at least in my hands. We find we can still get maximal expression from many promoters like trc with only 0.2 mM IPTG, and the response doesn't modulate well: it's either on or off. Growing at

Re: [ccp4bb] comparisons - views

2014-10-17 Thread Roger Rowlett
I've had a Gryphon for 2+ years and use it in an undergraduate environment. It's been trouble-free, and there are no instrument consumables, just blocks and trays. OK, I do have to feed it deionized water and a PCR tube of diluted protein for each set. It can set a 96-well tray in under 2

Re: [ccp4bb] Off topic: reformatting screens from Falcons into deepwell blocks

2014-10-01 Thread Roger Rowlett
The Art Robbins Scorpion has this function baked in the the operating software. When I was shopping for liquid handling robots for screen-making, only the Emerald Biosystems Opti-Matrix and the Scorpion were affordable for a small lab. The Scorpion can handle more solutions at one time.

Re: [ccp4bb] frozen pellet insoluble protein

2014-09-29 Thread Roger Rowlett
I have experience with some proteins that don't tolerate freeze-thawing very well. It's hard to say exactly what the physical chemistry of this is, but it probably relates to (1) aggregation due to high concentration or protein or salts during the freezing process as water is removed, and/or

Re: [ccp4bb] refine structure with mtz file in P212121 or P222?

2014-09-05 Thread Roger Rowlett
As always, look at the unit cell packing of your alternative solutions. In all likelihood one of these two solutions from Phaser should pack sensibly in the unit cell, and the other will not. You may get some sort of quasi-reasonable-looking electron density out of the wrong solution

Re: [ccp4bb] Off topic: Precast gels

2014-08-29 Thread Roger Rowlett
Define cheap. Several vendors offer SDS PAGE minigels for about $10 US a pop. I get mine from Novex. Roger Rowlett On Aug 29, 2014 2:22 AM, Theresa Hsu wrote: Dear all Would anyone knows of source of cheap precast SDS-page gels? Thank you.

Re: [ccp4bb] Exporting Omit Maps for use in Pymol using CCP4i FFT

2014-08-20 Thread Roger Rowlett
Did you enter a valid pdb filename for a coordinate file when selecting the option 'cover all atoms in pdb'? __ Roger Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University On Aug 20, 2014 3:54 PM, Wei Shi wrote: Hi all, I am

Re: [ccp4bb] Removing PEG3350

2014-08-20 Thread Roger Rowlett
Excellent references. PEG 3350 appears to be hydrodynamically equivalent to a 20 kD globular protein. So for efficient separation, your protein needs to be significantly larger than 20 kDa on a GEC column. In a centrifugal filter (which is very inefficient--you need many exchanges and

Re: [ccp4bb] Enigmatic electron density attached to Cys residue

2014-08-19 Thread Roger Rowlett
be a degradation product of PEG200? Did any MPD get into the mix? We have seen spurious oxidation of Cys to Cys-OH in thiol proteins, e,g, PDB 3UAO. Good luck. With that high quality ED, you'd think it would be easy to ID... Roger Rowlett Dear all, We are currently working on a small GTPase


2014-08-19 Thread Roger Rowlett
in the solution to minimize electrostatic interactions, e.g. 100-500 mM NaCl. Ultimately, your protein just may not be very soluble. That is potentially will ppt and maybe xtallize well at low concentration. Roger Rowlett On Aug 19, 2014 1:52 PM, Prashant Deshmukh prashantbiophys

Re: [ccp4bb] Zinc binding protein expressed from insect cells

2014-08-15 Thread Roger Rowlett
Harvey, Depending on the zinc-binding site, it may not bind Fe(II) at all. Zn(II) and Fe(II) have very different preferred ligand binding environments. For many zinc-metalloenzymes, substitution with Fe(II) would be difficult to impossible. In general, you will find it very difficult to make

Re: [ccp4bb] CC-half value ??

2014-08-14 Thread Roger Rowlett
Exactly. Aimless will give you suggested resolution cutoffs based on CC 1/2 in the log file. Roger Rowlett On Aug 14, 2014 5:04 PM, conan仙人指路 wrote: Hi Faisal, CC-half standard is valuable in evaluating the cut-off of highest resolution. Sometimes even if I/sigI

Re: [ccp4bb] Have everyone had a Scorpion Screen Builder or a Dragonfly screen optimizer?

2014-08-05 Thread Roger Rowlett
of coffee, about 10-15 minutes to dispense. No way we're going back to hand dispensing. Roger Rowlett Gordon Dorothy Kline Professor Colgate University On Aug 5, 2014 5:05 PM, Joseph Ho wrote: Dear all: We are interested in purchasing either a Scorpion Screen Builder from ARI

Re: [ccp4bb] packing test PHASER

2014-06-17 Thread Roger Rowlett
Increase the number of allowed clashes in Phaser, re-run it then look at the packing of the solution found and identify the source of the clashes. Possibilities for the clash issue include: 1. Wrong space group 2. Flexible loops or termini in search model not present or differently

Re: [ccp4bb] metal chelation

2014-05-19 Thread Roger Rowlett
The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators o An active site coordinated metal?

Re: [ccp4bb] metal chelation

2014-05-19 Thread Roger Rowlett
and advice. -Adam On May 19, 2014, at 12:55 PM, Roger Rowlett wrote: The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose

Re: [ccp4bb] PDB passes 100,000 structure milestone

2014-05-15 Thread Roger Rowlett
A logarithmic plot of cumulative entries to the PDB is approximately linear and shows a growth rate of about 15% per year. That means it doubles in size about every 5 years at current growth rate. Roger Rowlett On May 15, 2014 4:23 AM, Colin Nave wrote: From James's

Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement

2014-05-15 Thread Roger Rowlett
For P212121, I would put money on something divisible by 2 for the total molecules per asu. Anything from 6-12 might be likely. One of the early structures I worked on had 6 molecules per asu, which was darn near impossible to find using momomers (at the time). The way it was eventually solved

Re: [ccp4bb] metal ion coordination

2014-04-17 Thread Roger Rowlett
See below. Ceeers, ___ 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: On 4/17/2014 4:13 PM,

Re: [ccp4bb] Coot error

2014-04-15 Thread Roger Rowlett
Try yum install glib2-devel (as root). I haven't used CentOS/Fedora in a while, but I think this is the right package. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346

Re: [ccp4bb] About crystallization diffraction problem

2014-04-10 Thread Roger Rowlett
A couple of thoughts: * We have actually managed to grow quite a few crystals like this. Sometimes they are not single crystals, but stacks of mis-aligned plates that come apart easily when handled or subjected to osmotic stress. Sometimes these stacks give great-looking diffraction

Re: [ccp4bb] High Salt Cryo

2014-02-18 Thread Roger Rowlett
How about a short swish in well solution + 25-30% glucose? Doesn't take long to cryoprotect, just a quick sufrace coat. Sodium malonate? We just froze some really fragile crystals from 1.8 M sodium formate in 3 M sodium malonate and they held up really well. (Still didn't improve their

Re: [ccp4bb] suggestions for cryoprotectant

2014-02-06 Thread Roger Rowlett
Lots of choices. I usually try the crystallization solution + 30% glucose first. Glycerol or ethylene glycol are other possibilities here. Another possibility is 2.5-3.0 M sodium malonate at a similar pH. Roger Rowlett On Feb 6, 2014 11:40 PM, Deepak Thankappan Nair wrote

Re: [ccp4bb] off-topic: bug busting

2014-02-04 Thread Roger Rowlett
BeadBeater. Gentle, aerosol-free way to break 15-350 mL of cell paste (2-150 g wet packed cells). Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

2014-01-30 Thread Roger Rowlett
with mixing ratio, but will be easily repeatable. The actual pH of the final, magic solution can be directly measured if desired. Calculations will never be exactly right; pKa values are ionic strength dependent. Better to measure. Roger Rowlett On Jan 30, 2014 2:37 AM, sreetama das somon_

Re: [ccp4bb] Protein Purification Problem

2014-01-30 Thread Roger Rowlett
Do you really need to remove the NaCl? Some ionic strength is often necessary to stabilize proteins. Our routine purification buffers all contain at least 100 mM NaCl. This will not usually interfere with crystallization screening. To minimize the probability of aggregation, you need to (1)

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

2014-01-30 Thread Roger Rowlett
it, Hampton has a nice link to calculate volume of components while designing a tray as long as you tell it the concentrations. Nick From: Roger Rowlett Reply

Re: [ccp4bb] Lysis of E coli

2014-01-28 Thread Roger Rowlett
We do not have experience with this product. We use a BeadBeater. Can handle up to 25-30 g of wet packed cells in the medium beater jar. The large jar will handle maybe 3-5x that, but I've never had to go to that scale. ___ Roger Rowlett Gordon Dorothy Kline

Re: [ccp4bb] cryoprotection ideas for salt based condition

2014-01-22 Thread Roger Rowlett
Mahesh, Try 25-30% glucose. You can gradually add well solution + 37.5-40% glucose directly to the drop if your crystals are sensitive to changes in osmotic pressure. If your drop is evaporating too quickly, try working in the cold room or under oil to slow down evaporation. You can find our

Re: [ccp4bb] Phasing with Many Monomers/AU

2014-01-20 Thread Roger Rowlett
I agree. Searching with a larger unit is likely to be successful if you have a good idea of the structure of that larger unit. We had an example of a low homology (29% identity) MR situation with 8 subunits per ASU with twinned data. Not solvable with monomers. Solvable with a dimer search

[ccp4bb] ACA 2014 meeting--Session on engaging students in protein crystallography

2014-01-14 Thread Roger Rowlett
I am co-organizing (with Kraig Wheeler) a session at the 2014 American Crystallographic Association Meeting in Albuquerque, NM concerned with engaging undergraduate students in protein crystallography. I would like to encourage anyone who has involved undergraduate students in protein

Re: [ccp4bb] Cryo solution for crystals grown in magnesium formate

2013-12-16 Thread Roger Rowlett
Junyu, I haven't tried it personally with this particular solution, but I have found that 30% glucose can pretty much cryoprotect any condition I have tried it with. If necessary, add cryoprotectant solution (mother liquor + 30% glucose) gradually to minimize osmotic shock and potential

Re: [ccp4bb] Can Mathew's coefficient tell about a complex

2013-12-04 Thread Roger Rowlett
Looks like 1 or 2 protein units per ASU. Two seems most likely, but I have had a bunch of crystals with 67% solvent, too, so 1 is not out of the question if less probable. So try both with your favorite MR method and see what happens. Crystal packing in the MR solution will tell all.

Re: [ccp4bb] out of topic: liquid handling robot for optimisation

2013-11-29 Thread Roger Rowlett
, but of course this will be somewhat slow for accuracy. Software is easy to use--my undergraduates program and use it routinely. PEG blocks take 20-30 min, non-viscous blocks maybe 10 min. Roger Rowlett On Nov 29, 2013 5:12 AM, Valerie Biou wrote: Dear all, We are looking

Re: [ccp4bb] About protein precipitation problem during dialysis

2013-11-20 Thread Roger Rowlett
Right on target. Many proteins require some ionic strength in the solution to maintain solubility and prevent protein aggregation. Usually NaCl is used for this purpose. You can also use glycerol to enhance solubility, but this may interfere with crystallization if that's the next step. NaCl

[ccp4bb] lib32gfortran3 needed for 64-bit ccp4?

2013-11-11 Thread Roger Rowlett
Just ran into an error running SORTMTZ in CCP4-6.4.0. Looks like SORTMTZ requires 32-bit version. I get an error message of the missing library if I do not install lib32gfortran3 (32-bit fortran libraries). Should this still be necessary for the 64-bit CCP4? I'll keep this

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