Re: [ccp4bb] Bendability of DNA

You can try 3DNA, available at x3dna.org. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu Date:Wed, 31 Dec 2014 10:50:35 +0530 From:Gajanan Arbade gajanan_pbi...@diat.ac.in Subject: Bendability of DNA Hello CCP4 users, I

Re: [ccp4bb] workstation crystallography

with your favorite linux distribution. Aloha, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

Re: [ccp4bb] Incubator for crystallization

We use wine refrigerators. Peltier cooling, no compressor, no vibrations, cheap. They look nice too. But they can't cool to 4C. Aloha, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

[ccp4bb] Seeking software for Dynapro 99 DLS

Hi, Can someone help me with obtaining the software to operate a Dynapro 99 DLS instrument? Wyatt no longer provides software for this model. Newer versions of Dynamics are not compatible with the Dynapro 99. Thank you! Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor

Re: [ccp4bb] Coot Performance on Intel HD Graphics 5000

Coot and PyMol run fine on my HD4000. I believe I also have an HD3000 in the lab that is going strong. I look forward to running Coot on my Android before long. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

Re: [ccp4bb] Determining concentration of membrane protein

Hi Raji, There are also some proprietary stains such as the 660 nm (can't they think of a better product name?) stain from Pierce that are detergent compatible. I used this briefly with success when comparing against Abs 280 nm. Ho Ho Leung Ng University of Hawaii at Manoa Assistant

Re: [ccp4bb] Room temperature data collection

to it, oils are very easy to work with. You can work with your oil mounted crystals at room temperature or flash freeze for cryo-collection. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

Re: [ccp4bb] Best compounds for heavy atom soaks

A favorite resource is Bart Hazes' web page on heavy atom derivatives. http://homepage.usask.ca/~pag266/bart-hazes.html Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

Re: [ccp4bb] Off topic: Gel filtration of membrane protein

If your buffer can't go through a 0.2 micron filter easily, you shouldn't run it through your FPLC. Detergent purity may be an issue. I also experienced problems filtering a buffer containing CHAPS from vendor X. When I switched to Anatrace CHAPS, no more clogging. Ho Ho Leung Ng

Re: [ccp4bb] Double stranded DNA concentration estimation

Hello Appu, Small amounts of strongly UV absorbing contaminants can throw off your measurements. If you want to be very accurate, try a fluorescent dye specific for double stranded DNA such as PicoGreen. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department

[ccp4bb] Windows 8?

Hello, Are there any issues with crystallography related software on Windows 8, especially with PyMol or Coot? Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

Re: [ccp4bb] Membrane Protein Optimisation

Hello Rhys, I suggest using an assay such as that described in Anal. Biochem. 336:117 to measure the amount detergent in your concentrated samples. I found this assay accurate and easy to use with DDM. It should work with b-OG too. Good luck, Ho Ho Leung Ng University of Hawaii at Manoa

Re: [ccp4bb] gelification of a pure protein

in the protein buffer. I was not able to crystallize that receptor. I like to think we stumbled on some kind of cool receptor oligomerization phenomenon. I wish I had done more to characterize the gel instead of cursing the lack of crystals. Ho Ho Leung Ng University of Hawaii at Manoa Assistant

Re: [ccp4bb] Anyone has experience with digesting membrane protein by precession protease

. I have also heard from a few other researchers that GFP fusion can sometimes promote aggregation. I'd appreciate hearing from others how often they have encountered GFP-mediated aggregation, whether with membrane or soluble proteins. Ho Ho Leung Ng University of Hawaii at Manoa Assistant

Re: [ccp4bb] Need specific molecular replacement test cases

Hi Raji, For case #2, an example is calmodulin, which displays remarkable acrobatics across crystal forms, and +/- ligand. I look forward to reading about the extreme cases that satisfy case #1 on the list! Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department

Re: [ccp4bb] CCP4BB Digest - 3 Feb 2013 to 4 Feb 2013 (#2013-35)

Hi Dave, My experience is that students learn much better when they work with colored proteins or crystals. Myoglobin sounds good, but I haven't worked with it before. I worked previously with a GFP variant, and that was challenging to grow good crystals. Ho Ho Leung Ng University

Re: [ccp4bb] Off-topic: Best Scripting Language

I encourage trainees to learn a programming language that they will help their careers beyond their short time in my lab. Many or most of them will not continue in structural biology or even science. For the moment, I am pushing python even though I am minimally literate in it myself. They

[ccp4bb] the lysozyme of membrane proteins?

leading candidate is aquaporin AqpZ from E. coli. I am planning to express the membrane protein as a GFP fusion so students can easily follow it through the course of the labs. Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

[ccp4bb] Postdoctoral Fellow, Protein crystallography - University of Hawaii

Applications are invited for a postdoctoral position in the lab of Ho Leung Ng, Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA. Multiple projects are available involving antibody complexes, GPCRs, structure based drug design, natural products drug discovery, protein

Re: [ccp4bb] Purify non-stable protein

In addition to the other very good suggestions, you could consider using a purification tag at both the N- and C-termini of your protein to only pull out full length protein. I've had success with Flag+His and MBP+His. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department

Re: [ccp4bb] Optimizing xtals conditions

Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

Re: [ccp4bb] Heme incorporation in expressed protein

I was able to express a heme protein by inducing and expressing at room temperature and using a promoter weaker than T7 (can't remember the exact one right now). The key was to slow down the rate of protein production to allow heme incorporation. You might try using less IPTG too. Ho Leung Ng

[ccp4bb] Akta vs HPLC

the machines, columns, and proteins happy? Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

Re: [ccp4bb] No subject given

Hello Ruby, I would start by verifying the functional status and monodispersity of your protein. If it appears that you have a good protein sample, I suggest screening with a more concentrated protein solution. What protein concentrations have you been you working with? Ho Ho Leung Ng

Re: [ccp4bb] Suggestions for solving a structure with 8-10 copies per asymmetric unit

. With Phaser, you'll probably want to loosen the acceptable number of packing clashes. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu On Mon, Apr 30, 2012 at 1:00 PM, CCP4BB automatic digest system lists...@jiscmail.ac.uk wrote: Date:    Mon

Re: [ccp4bb] Crystal handling

Have you tried the MiTeGen Micromesh? They're my favorite for handling tiny crystals. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu On Sat, Apr 7, 2012 at 1:00 PM, CCP4BB automatic digest system lists...@jiscmail.ac.uk wrote: Hi

Re: [ccp4bb] Who is using 64-bit Linux?

Roger, My lab is using 64 bit distros of SUSE and Linux Mint and hasn't had any compatibility issues that I can recall. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu Date:Tue, 3 Apr 2012 15:57:40 -0400 From:Roger Rowlett

Re: [ccp4bb] DDM

Actually, DDM is the most successfully used detergent for membrane protein crystallization. See Newstead et al, Protein Sci. 17:466. But yes, the rule of thumb is that detergents that form smaller micelles give better diffracting crystals, but are more destabilizing. Ho Ho Leung Ng University

[ccp4bb] Postdoctoral position - University of Hawaii

first author publication. The fellow must be able to work independently and enjoy mentoring students. Applicants should send their CV and contact information of three referees by email to me (hng @ hawaii.edu). Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry

Re: [ccp4bb] Aggregated protein for crystallization

I should have been more clear. If your protein is insoluble aggregate, you can use crystal screen results to get an idea of what buffer conditions favor solubility (and hopefully monodispersity). An example is described nicely in Collins et al, Acta Cryst F 61:1035. Ho Ho Leung Ng University

Re: [ccp4bb] Aggregated protein for crystallization

buffer conditions. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu

Re: [ccp4bb] Manually setting 96 wells plates with lower volume samples!

up. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry holeung...@hawaii.edu From:xaravich ivan xaravich.i...@gmail.com Subject: Manually setting 96 wells plates

Re: [ccp4bb] Magnetic anti-flag beads

If it's the M1 antibody, you can elute with EDTA + Flag peptide. You could also try a little higher pH like 3.5. Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry holeung...@hawaii.edu On Thu, Nov 17, 2011 at 2:00 PM, CCP4BB automatic digest system

Re: [ccp4bb] Protein-Protein Complex Screening

There is at least one paper describing the success of PEG precipitants for complexes, but I can't find the reference right now. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry holeung...@hawaii.edu

Re: [ccp4bb] Vivaspin vs Amicon Ultra

type. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry holeung...@hawaii.edu

Re: [ccp4bb] CCP4BB Digest - 13 Jan 2011 to 14 Jan 2011 (#2011-16)

Hi Gloria, I used to use the Tev construct described in Cabrita et al (Prot. Sci. 16:2360) which is more stable and soluble. ho

Re: [ccp4bb] update--degradation of MBP fusion protein

Hi Jerry, Try parameters that slow the rate of protein production, such as expressing at lower temperature, using less IPTG/inducing agent, or using a weaker promoter. Good luck, Ho

Re: [ccp4bb] crystal growth

If I remember correctly, NaF forms octahedral crystals. Be sure to check for salt crystals in your reservoir well. ho

Re: [ccp4bb] How to detect the concentration of detergent?

I like the phenol-sulfuric acid colorimetric assay (Anal Biochem. 2005 Jan 1;336(1):117-24). Easy, very linear, no special ingredients/equipment required. In agreement with their paper, I also found that DDM passes through the 100 kD concentrator membranes. Ho

Re: [ccp4bb] CCP4BB Digest - 4 Sep 2010 to 5 Sep 2010 (#2010-243)

Hello Hari, It looks like Addgene has both pCW-LIC and pCWori available. ho

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

Some other things to try: 1. Co-crystallize with a ligand 2. crystallization with lipid cubic phase or bicelles 3. limited proteolysis to define a rigid core ho - Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition

Re: [ccp4bb] Problems in purification

I assume you are trying to do a non-denaturing prep. Have you run your sample on a size exclusion column to see if it is aggregated? If it is aggregated, it can stick to a lot of contaminating proteins which will be difficult if not impossible to separate. ho On Thu, Aug 26, 2010 at 8:24 AM,

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

Hi Pascal, I suspect the protein is aggregating in the presence of FosCholine. In addition to the suggestions made by others, you can also try changing the salt concentration or including additives like glycerol in your FosCholine buffer. This can make an enormous difference in the stability

Re: [ccp4bb] Beginning crystallography text

Back when I was a graduate student, my favorite book was Drenth. However, that book was never a favorite with most students, who preferred Crystallography Made Crystal Clear. I also think the Blow book is good. I'm not familiar with the newer books written by our mailing list colleagues. ho

Re: [ccp4bb] UV microscope

I used a Korima a few times and didn't like it. Poor image quality and you have to worry about nuking your crystals with UV. However, I haven't tried any other UV microscopes to compare. For most purposes outside of high throughput imaging, I'd rather just shoot the mystery crystals with xrays.

Re: [ccp4bb] Converting cif-files into pdb-files / reading cif -files into coot

Don't you worry, there's a lifetime of learning opportunities. I used to spend almost as much time helping senior PIs with format conversion as new students. Got to love the pdb ho --- From:

Re: [ccp4bb] question - GFP fusion - cleavage sites

What did you see on your ion exchange and gel filtration chromatographs? ho

Re: [ccp4bb] Cryo Vs crystal size

Like others have pointed out, I've often found very large crystals ( 0.4 mm) to not diffract as well, either due to growth defects or poor cryoprotection. How large are the crystals you're talking about? You could try chipping a piece off your large crystal and see how well that diffracts. ho

Re: [ccp4bb] crystals of 1D

Have you tested how well they diffract? You should do that first. Sometimes small, ugly looking crystals can give good data. ho

Re: [ccp4bb] activation of thiol group

Hello Deepak, What pH are your crystals at? Also, you need to check whether your atomic arrangement has reasonable geometry for hydrogen bonding in addition to the interatomic distances. ho -- Date:Mon, 29 Mar 2010 12:11:59 +0800 From:Deepak Oswal

Re: [ccp4bb] TCEP effect on protein

Does your SDS-PAGE loading buffer contain a reducing agent like beta mercaptoethanol? That could be responsible for the difference between your SDS-PAGE and HPLC results. ho On Fri, Mar 26, 2010 at 5:01 PM, CCP4BB automatic digest system lists...@jiscmail.ac.uk wrote: There are 5 messages

Re: [ccp4bb] how to improve resolution

My interpretation of hollow crystals is that the crystals are growing too fast. I've had success with similar looking crystals by slowing crystal growth. You can try lower temperature, protein concentration, or precipitant. In my case, I found success by trying different concentrations of NaCl in

Re: [ccp4bb] Crystal rescue

Hi Zhiyi, I think the easiest way is to mount your crystal in an air-impermeable, viscous oil like Paratone in a loop. Of course, your crystals may not tolerate oil. ho

Re: [ccp4bb] Purchase of Protein Samples

Hello Stephen, Of course it depends on what proteins you want. You can buy some easily crystallizable proteins from Hampton Research. Some purified proteins (proteases, lysozyme, calmodulin, etc) can be purchased from Sigma. Or are you looking for custom expression and purification? ho

Re: [ccp4bb] smeared spot in diffraction

In addition to the good suggestions already posted, I also suggest: 1. Exploring how your cryoprotection is done in addition to screening chemical conditions. For example, you may want to test longer equilibration in cryoprotectant, stepwise increase in cryoprotect., very fast swish through

Re: [ccp4bb] CCP4BB Digest - 12 Jan 2010 to 13 Jan 2010 (#2010-12)

Hi Nick, In the past, I've successfully expressed a 130 kd eukaryotic protein in large quantities in plain BL21. I don't think size per se is a major problem for expression although I've heard others say they had problems with heterogeneous termination. I'd try the standard repertoire of

Re: [ccp4bb] contaminants in PEG - references

Zhang and Tanner, 2004, Detection of L-lactate in polyethylene glycol solutions confirms the identity of the active-site ligand in a proline dehydrogenase structure, Acta Cryst D. 60:985

Re: [ccp4bb] To get the crystal faster...

I can't remember if someone has already suggested this. You can dissolve some of your crystals and ask your favorite mass spec. lab to check if your protein has been oxidized, proteolyzed, etc. ho Confometrx

[ccp4bb] mammalian cell culture on IMAC

This article discusses this effect in E. coli lysates. Please let us know if you find something that works well with insect/mammalian lysates or secreted proteins! Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates

Re: [ccp4bb] Reducing Agent Tips?

Yes, try the TCEP. You can also try alkylating agents like iodoacetamide. Also consider mutating away your cysteines. Ho ConfometRx

Re: [ccp4bb] Cold Cabinet Vs Fridge, FPLC

I know several labs that keep their FPLCs at room temperature. Maybe the cold cabinet isn't necessary? ho

Re: [ccp4bb] moelcular replacement with large cell

Are you sure you're using the right space group? For example, what does phenix.xtriage suggest for your space group? Ho Confometrx

Re: [ccp4bb] high number of lysines

Hello Amit, 1. You may have to dramatically increase your protein concentration to get into the crystallization range. What is the maximum solubility of your protein? What protein concentration are you currently working with? 2. Try the UCLA surface entropy reduction server

Re: [ccp4bb] MAD phasing

You need to try alternative space groups, such as P6522, which has the same extinctions and merging statistics as P6122. What do the maps look like coming out of SOLVE/RESOLVE? Ho UC Berkeley

Re: [ccp4bb] How to improve crystal which is twinning?

Hello Annie, How effective have you found using sparse matrix screens as additives vs. traditional additive screens? I've tried this only a few times without success and would like to hear from someone with more experience. Thanks! Ho UC Berkeley

Re: [ccp4bb] How to improve crystal which is twinning?

Try things that affect nucleation and kinetics of crystallization, such as seeding or crystallization at different temperatures. As Morten suggested, try different crystallization methods such as switching between hanging/sitting drop and microbatch. Screen protein concentration and

Re: [ccp4bb] Refining partially occupied DNA on top of itself

Hi Bryan, Can't you choose an asymmetric unit that includes two protein molecules and the entire non-palindromic DNA? ho

[ccp4bb] Fwd: Aussie synchrotron sports sponsorship - very funny

I hope this helped our Australian friends get more funding! Ho The TV show 'Thank God You're Here' goes to the synchrotron! http://www.youtube.com/watch?v=N_zbySqumaAfeature=related

Re: [ccp4bb] problem in transformation of pqe 30 clone

Hello Artem, We express almost all our proteins in BL21 derivatives. It sounds like you've worked with many proteins that express/behave better in XL1-Blue? ho UC Berkeley - XL1-Blue is a

Re: [ccp4bb] Cryo-protectant

Your mother liquor is already very close to being a cryoprotectant. I think adding 5% ethylene glycol, glycerol, etc. is enough. Too much may damage your crystals. In addition, you can also try: 1. Grow new crystals in your current crystallization condition + 5% cryoprotectant. 2. Increase the

Re: [ccp4bb] protein purity in SAXS

Since the SAXS signal comes from the size of your scattering particles, make sure you don't have large non-protein particles in your sample (dust or micelles, for example). The SIBYLS web site has additional warnings about including detergents in your sample. ho UC Berkeley

Re: [ccp4bb] weak density for the missing part

Hello Raja, Are you sure your smaller protein is in your crystal? Have you run a gel or performed mass spec on dissolved crystals? ho UC Berkeley

Re: [ccp4bb] Halide soaking

Around here, I would guesstimate the success rate to be in the 5-10% range. Still, I always try it as it's so easy. I don't think longer soak times will usually do much to increase occupancy. Instead, try higher halide concentrations. I also like to try monovalent cations (Rb, Cs) but

Re: [ccp4bb] Design Constructs

Hello Hari, The general rule is to truncate/delete residues that are predicted to be disordered, for example, by secondary structure prediction or homology modeling. You do have to be careful as there may be functionally important, conserved, structured loops that you want to retain.

Re: [ccp4bb] selenomethionine labeling

Hello Jerry, We use the Van Duyne et al feedback inhibition method with BL21 and have never had a problem. ho UC Berkeley -- Date:Thu, 26 Mar 2009 14:58:57 -0700 From:Jerry McCully for-crystallizai...@hotmail.com Subject: selenomethionine labeling

Re: [ccp4bb] Purification

You can try using affinity tags on both the N- and C-termini of the protein, eg. MBP on N and His on C. ho Date:    Thu, 19 Mar 2009 23:53:14 + From:    Kn Ly kn...@auckland.ac.nz Subject: purification Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli.

Re: [ccp4bb] How to refine a solution obtained by molecular replacement

I've found CNS's simulated annealing composite omit maps to be very useful in situations like this to avoid phase bias. RESOLVE's prime and switch offers similar functionality, but I've had less experience with it. ho UC Berkeley

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

Most of the poorly cleavable fusion proteins (usually MBP-TEV) that I've seen turned out to be solubly aggregated. ho UC Berkeley -- Date:Fri, 27 Feb 2009 07:23:43 -0500 From:Stephen Weeks stephen.we...@verizon.net Subject: Re: Off topic: Mammalian gene

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

Some useful tips to try can be found at http://www.embl-hamburg.de/services/protein/production/expression/optimising_exprlevels.html I've had a recent case where an untagged protein (part of a complex) was not expressed at all but expressed well when tagged at the N-terminal with His6 or MBP.

[ccp4bb] include or exclude overloads

-Leung Ng wrote: Can you elaborate on the effects of improper inclusion of low resolution (bogus?) reflections? Other than rejecting spots from obvious artifacts, it bothers me to discard data. But I can also see how a few inaccurate, very high intensity spots can throw off scaling. I

Re: [ccp4bb] CCP4BB Digest - 12 Feb 2009 to 13 Feb 2009 (#2009-45)

Hi Clemens, Can you elaborate on the effects of improper inclusion of low resolution (bogus?) reflections? Other than rejecting spots from obvious artifacts, it bothers me to discard data. But I can also see how a few inaccurate, very high intensity spots can throw off scaling. ho UC

Re: [ccp4bb] 2D

Along the lines of Jeroen's suggestion, we've enjoyed success with surface entropy reduction mutations to alter crystal contacts. UCLA has an SER analysis server at: http://nihserver.mbi.ucla.edu/SER/ Ho UC Berkeley

Re: [ccp4bb] Need help for solving a tough problem of phasing

Hello Jian Wu, Because the packing does not allow room for your Sub1 and Sub2 domains, I suspect your MR solutions are not correct. It can be surprisingly difficult to tell from your electron density due to phase bias. I've found both simulated annealing omit and Resolve prime and switch