Re: [ccp4bb] Bendability of DNA

[Ho Leung Ng]

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 
Subject: Bendability of DNA

 Hello CCP4 users,



 I am working with some DNA binding proteins. Can someone explain me how to
measure/calculate the bendability of a DNA molecule after binding to
proteins and foldabilty of protein molecule?


It will be good if you mention any reference(s).


 Thanks in adv.

-Gajanan
__
Gajanan K Arbade

Research Scholar

Defence Institute of Advanced Technology (DIAT)

Pune Maharashtra, India

Pin Code-411025

Contact No. Lab.+ 91-20-24304377

 Mob: 08698198016

Re: [ccp4bb] B-factor blurring

[Ho Leung Ng]

Another useful reference:

Liu C & Xiong Y (2014). Electron Density Sharpening as a General
Crystallographic Technique. *J. Mol. Biol.* 426, 980-993.
http://www.ncbi.nlm.nih.gov/pubmed/24269527

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

Re: [ccp4bb] Incubator for crystallization

[Ho Leung Ng]

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

Re: [ccp4bb] workstation crystallography

[Ho Leung Ng]

The more recent integrated graphics chips, starting from the Intel HD3000,
have adequate performance for crystallography. Really, most crystallography
applications are less computationally demanding than video games now. I
think the most important issue is to make sure your hardware plays nicely
with your favorite linux distribution.


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

[Ho Leung Ng]

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, Department of Chemistry
h...@hawaii.edu

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

[Ho Leung Ng]

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] Trouble cleaving SUMO tag off of membrane protein

[Ho Leung Ng]

Hi Raji,

  Your membrane protein should be in micelles as a protein-detergent
complex. The detergent belt buries much of the protein surface area and can
make a lot of biochemistry experiments less efficient.

  I've never worked with SUMO tags before, but if cleaving them is like
with other tags, I suspect the cause of your problems is due to your
membrane protein aggregating, which they love to do. Have you screened for
aggregation or biological activity in different detergent buffers and
expression conditions yet?

  You may get lucky and improve cleavage by changing your buffer to
decrease aggregation. Try very low salt, high salt (up to 1M), and glycerol.

 If your protein is well behaved, you may have more success cleaving
your tag by increasing the linker length.


Ho


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


Date:Sun, 16 Feb 2014 10:58:12 -0500
> From:Raji Edayathumangalam 
> Subject: Trouble cleaving SUMO tag off of membrane protein
>
> Hi Everyone,
>
> After several attempts to cleave the SUMO tag off my membrane protein under
> various conditions (different reducing agents, enzyme-to-substrate ratios,
> etc.) and after reading the manual and troubleshooting guide, I'm reaching
> out to the ccp4bb community.
>
> Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express
> protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or
> 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am
> currently using an enzyme-to-substrate molar ratio of 1-to-15-20.
>
> Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved
> protein and 50% tagged protein. With buffer containing 2mM bME, I get about
> 30% tag-cleaved protein and 70% tagged protein.
>
> Couple of things:
> (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the
> same batch of Ulp1 works to 100% completion.
> (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the
> SUMO-tagged control soluble protein.
> (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with
> 4C, a protocol that I have used successfully in the past with SUMO-tagged
> soluble proteins.
>
> Although membrane proteins supposedly form a protein-detergent complex, I
> wonder if some of my protein is in micelles and if the random orientation
> of my SUMO-tagged protein in micelles may be the cause for incomplete
> digestion. I've also suspected that some of my membrane protein may be
> misfolded and oligomeric/aggregated, making the cleavage site inaccessible
> to the protease.
>
> But suppose the above explanations are not the problem in my case and that
> it's a technical issue and I am missing something very simple. Therefore, I
> am planning to set up more reactions ramping up the ratio of
> enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since
> I need to rebind the cleaved mixture to His-affinity resin) and decreasing
> the NaCl concentration to 100mM or lower (although 250mM NaCl did not
> interfere with cleavage of control protein).
>
> Have folks working with SUMO-tagged membrane protein encountered similar
> problems? I am purifying membrane protein from 30L bacterial culture and
> the yields are not all that great. So, if possible, I'd like to get the
> cleavage reaction to completion so that I don't have to suffer a 50% loss
> of protein at this step. I have a construct for my membrane protein without
> a SUMO tag and the expression is abysmal.
>
> Thanks very much for your time and suggestions!
> Raji
>
> --
> Raji Edayathumangalam
> Instructor in Neurology, Harvard Medical School
> Research Associate, Brigham and Women's Hospital
> Visiting Research Scholar, Brandeis University
>

Re: [ccp4bb] Determining concentration of membrane protein

[Ho Leung Ng]

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 Professor, Department of Chemistry
h...@hawaii.edu


Date:Thu, 13 Feb 2014 10:06:12 -0500
> From:Raji Edayathumangalam 
> Subject: Determining concentration of membrane protein
>
> Dear CC4BBers,
>
> I am trying to figure out what is the best way to determine the protein
> concentration of my membrane protein. My purified membrane protein is in
> 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).
>
> After reading the friendly manuals and searching online, I've learned that
> detergents interferes with assays like Bradford but can't find good
> descriptions of what works best. For now, I am trying to estimate
> concentration from absorbance at 280nm and using molar extinction
> coefficients based on aromatic amino acids, but again suspect detergent
> interference. I would like to know what other folks working on membrane
> proteins are doing.
>
> Thanks very much.
> Raji
>

Re: [ccp4bb] Room temperature data collection

[Ho Leung Ng]

I recommend using an air-impermeable oil like Paratone over the hassle of
capillaries. People often say that oil kills their crystals, but in my own
experience, that rarely happens. I think it is more likely due to
mishandling as working with oil does require some practice.

Once you get used 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

[Ho Leung Ng]

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

[Ho Leung Ng]

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


We are trying to purify a membrane protein using different detergents (DDM,
OG etc.). We have tried using 1mM DDM in 20mM Tris, 5mM NaCl and 5%
glycerol buffer to purify the protein. however, we are facing problems in
running the buffer in 16/60 Superdex 200 pg gel filytration coloumn using
AKTA Explorer. The entire machine is in a cold cabinet. The buffer was also
kept at constant slow stirring, thinking that it might be getting
precipitated, which we are not able to see. Still the back pressure is very
high and the in-line filter keeps clogging. We have filtered the buffer
through a 0.2 micron filter, which too was very difficult. the

Has anyone faced a similar proble? Or is there a way that buffers with
detergents are supposed to be made? Or are there any particular coloumns
meant for such runs.

Re: [ccp4bb] Double stranded DNA concentration estimation

[Ho Leung Ng]

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 of Chemistry
h...@hawaii.edu


> Deal CCP4 member's
>   Sorry for asking Off topic question.
> Please correct me if i am doing wrong measurements. I am determining the
> double stranded concentration using the absorbance spectrophotometer
> reading at 260A and  extinction coefficients for individual NMPs . But
> protein to DNA stoichiometry is coming really off in ITC. I am doubting
> weather i am using right method to determine the DNA concentration
> accurately or i am doing some wrong calculation. I am calculating the DNA
> extinction coefficients by simply multiplying the total number of
> individual nucleotide by it extinction coefficients which are as follow
> A = 15.4 mM-1 cm-1
> C = 7.4 mM-1 cm-1
> G = 11.8 mM-1 cm-1
> T = 9.6 mM-1 cm-1
>
> The formula which i am using to calculate the concentration is
>   DNA concentration(M)=   ( Abs@260-Abs@330)/Molar extinction
> coefficients of DNA
> I am multiplying this by dilution factor.

[ccp4bb] Windows 8?

[Ho Leung Ng]

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

[Ho Leung Ng]

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
Assistant Professor, Department of Chemistry
h...@hawaii.edu

Re: [ccp4bb] gelification of a pure protein

[Ho Leung Ng]

Hi Pascal,

 Is this a membrane protein? I worked on a GPCR that gelled upon
high concentration in a detergent/cholesterol solution. It was
reversible upon dilution and ran as a monomer by size exclusion
chromatography. Detergent assays showed that detergent was not being
concentrated 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 Professor, Department of Chemistry
h...@hawaii.edu

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

[Ho Leung Ng]

Hello Qiangmin,

  Many membrane proteins like to aggregate in detergent, which
will prevent efficient site specific proteolysis. If you have not
already done so, I suggest running a size exclusion column first to
verify that your protein is monodisperse in your detergent and buffer
of choice.

  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 Professor, Department of Chemistry
h...@hawaii.edu


On Mon, Apr 8, 2013 at 1:01 PM, CCP4BB automatic digest system
 wrote:
> Date:Tue, 9 Apr 2013 01:22:36 +0800
> From:Qiangmin Zhang 
> Subject: Anyone has experience with digesting membrane protein by precession 
> protease
>
> Hello everybody,
> I just purified a membrane protein tagged with GFP, which has a cleavage site 
> of precession protease. And I got a problem with removing the GFP tag by 
> precession protease (1mM DTT and 1 mM EDTA were included in the buffer). It 
> can not cut my protein. I have already tried to digest it in different 
> detergent like DDM and C12E8 (also different concentration for detergent like 
> lowering the detergent to 1.1 x cmc since a recent science paper lowered the 
> detergent to this level and got it worked). I know this depends on the 
> different proteins. I am wondering if anyone has this experience in digesting 
> membrane protein by precession protease. Any suggestions are appreciated. 
> Otherwise I might have to go back to just his-tag if there is no trick for 
> that. Thank you so much in advance.
> All the best
> Qiangmin Zhang

Re: [ccp4bb] Need specific molecular replacement test cases

[Ho Leung Ng]

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 of Chemistry
h...@hawaii.edu


On Fri, Mar 8, 2013 at 2:00 PM, CCP4BB automatic digest system
 wrote:
> Date:Fri, 8 Mar 2013 14:38:01 -0500
> From:Raji Edayathumangalam 
> Subject: Need specific molecular replacement test cases
>
> Hello Everyone,
>
> I am looking for two specific test cases (below) and appreciate anyone
> pointing me to known structures/examples for the same.
>
> (1) For a successful case of molecular replacement in which the search
> model has an overall sequence identity to the target in the twilight zone
> or worse (25% or less).
>
> (2) A case in which the search and target models share 80-100% sequence
> identity but where conformational changes in the target relative to the
> search model prevented a successful MR solution.
>
> Many thanks.
> Raji
>
> --
> Raji Edayathumangalam
> Instructor in Neurology, Harvard Medical School
> Research Associate, Brigham and Women's Hospital
> Visiting Research Scholar, Brandeis University

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

[Ho Leung Ng]

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

Re: [ccp4bb] SeCys usage for SAD

[Ho Leung Ng]

You might try mercury soaks instead. Might give you 15 minute answers to your 
questions.


HNY!

Ho

Sent from my iPad

On Dec 26, 2012, at 4:00 PM, CCP4BB automatic digest system 
 wrote:

> There is 1 message totaling 34 lines in this issue.
> 
> Topics of the day:
> 
>  1. SeCys usage for SAD
> 
> --
> 
> Date:Wed, 26 Dec 2012 12:33:02 -0600
> From:Linda Olson 
> Subject: SeCys usage for SAD
> 
> Dear All,
> 
> I have read a recent paper by Salgado et al about using non-auxotrophic 
> E.coli to incorporate SeCys into recombinant protein for phasing purposes.  
> Does anyone have a source for Selenocysteine?  I have also seen a paper by 
> Schaefer et al which uses nitric acid treated elemental Se for a sulfur 
> surrogate to generate Se-labeled protein.  Has anyone else tried this?  My 
> proteins are rich in Cys and tend to lack Met so the prospect of labeling cys 
> is very attractive.
> 
> Thanks,
> 
> Linda
> 
> Linda Olson, PhD
> Research Scientist II
> Dept Biochemistry
> Medical College of Wisconsin
> 8701 Watertown Plank Rd
> Milwaukee, WI 53226
> 
> phone: 414-955-8545
> fax:  414-456-6510
> _
> 
> --
> 
> End of CCP4BB Digest - 24 Dec 2012 to 26 Dec 2012 (#2012-363)
> *

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

[Ho Leung Ng]

 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 should learn a "modern" programming
language that is widely used beyond my subdiscipline. Python will
probably help them get a job more than Fortran.


Ho

[ccp4bb] the lysozyme of membrane proteins?

[Ho Leung Ng]

Hello,

  I am developing an undergraduate biochemistry lab class and
would like to incorporate experiments with membrane proteins. Does
anyone have suggestions on membrane proteins that are relatively easy
to express, purify, and assay? Bonus points for crystallizable! At the
moment, my 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

[Ho Leung Ng]

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 engineering, and developing methods
for membrane protein crystallization. The fellow will also be expected
to come up with her own project ideas. The laboratory benefits from
collaborations with industry and the medical school to pursue broad,
interdisciplinary approaches to challenging structural biology
problems.

We seek a highly motivated scientist with extensive experience in
cloning, protein expression, purification, characterization, and
crystallography. Expertise with multiple expression systems including
E. coli, yeast, and cell free systems is highly valued. Experience
with membrane proteins will be very helpful.

Applicants must have a Ph.D. and at least one first author
publication. The fellow must be able to work independently and enjoy
mentoring students.

Applicants should send their CV and the contact information of three
referees by email to Ho Leung Ng (hng @ hawaii.edu).

Re: [ccp4bb] Purify non-stable protein

[Ho Leung Ng]

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 of Chemistry
h...@hawaii.edu


On Tue, Aug 21, 2012 at 1:00 PM, CCP4BB automatic digest system
 wrote:
>
> Date:Tue, 21 Aug 2012 21:15:15 +0100
> From:Theresa Hsu 
> Subject: Purify non-stable protein
>
> Dear all
>
> I made deletion mutation of a stretch of 20 amino acids on my protein. I can 
> purify and crystallize wild type protein but not the mutated. Mass spec on 
> gel separated protein shows degradation of mutant losing about another 150 
> amino acids. Is there any way of purifying this non-stable protein? I know 
> nature has designed proteins to be stable.
>
> All steps are done at 4 C and protease inhibitor added during cell lysis for 
> both proteins.
>
> Thank you.

Re: [ccp4bb] Optimizing xtals conditions

[Ho Leung Ng]

Hello Christine,

 First, you want to make sure those crystals are not salt.
Crystals that appear only after a very long time are often salt
crystals that form as your drop dries out. Check diffraction to see if
it's the real thing before you invest time optimizing crystallization.


Ho

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

Re: [ccp4bb] Heme incorporation in expressed protein

[Ho Leung Ng]

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


On Mon, Jul 16, 2012 at 1:00 PM, CCP4BB automatic digest system
 wrote:
>
> Date:Mon, 16 Jul 2012 11:15:59 +0530
> From:Biswajit Pal 
> Subject: Heme incorporation in expressed protein
>
> Dear all,
> Sorry for non-crystallography related question.
> We are trying to express an eukaryotic heme protein in E. coli. We are able 
> to express it in soluble form. When we use 5-Aminolevulinic Acid, E. coli 
> becomes brown. However, after cell lysis the soluble protein contains no heme 
> and the pellet is brown. If we extract the pellet with detergent (Triton 
> X-100 and Tween-20) the color comes in the supernatant, but there is no 
> protein of our interest. These eventually indicate that the protein and heme 
> are getting expressed/synthesized, but heme is not getting incorporated in 
> the expressed protein. We would like to get this protein in heme-bound 
> holo-form.
> Any suggestion to trouble-shoot this problem would be highly appreciated. 
> Replies can be sent to me directly and I will post a summary on a later date.
> Thanks in advance,
> Sincerely yours,
> Biswajit Pal
> CCMB, Hyderabad, India

[ccp4bb] Akta vs HPLC

[Ho Leung Ng]

Hello,

 My Akta Purifier is being repaired, and I'm thinking about borrowing a
colleague's HPLC in the interim. What makes the Aktas different from HPLCs?
I've used HPLCs for purifying small molecules and peptides but not
proteins. Anything I should be careful about regarding keeping 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]

[Ho Leung Ng]

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


On Fri, May 4, 2012 at 1:00 PM, CCP4BB automatic digest system
 wrote:
> Date:    Fri, 4 May 2012 12:39:42 +0530
> From:    ruby singh 
> Subject: 
>
> Dear all,
>
> Im a PhD student who started working on Protein crystallization. Its been
> years im trying to get any crystal of that protein. I have tried all
> crystallization screens available.
> but no results. My protein is highy thermostable(till 80C) and pH stable(1
> to 13)...plz if anyone is having any idea or trick let me knw.

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

[Ho Leung Ng]

When searching for multiple molecules/ASU, you need to be careful with
how the software handles packing. Small but acceptable clashes can
accumulate and cause the searches to fail. I suggest using a highly
trimmed as well as a poly-alanine model. I've had success with both
epmr and Phaser. 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
 wrote:
> Date:    Mon, 30 Apr 2012 15:41:54 +
> From:    "Ke, Jiyuan" 
> Subject: Suggestions for solving a structure with 8-10 copies per asymmetric 
> unit
>
> Dear All,
>
> I have a question regarding solving a crystal structure by molecular 
> replacement. It is a single protein with a molecular weight of 25.5 kDa. The 
> cell dimension is rather big from the diffraction data ( 90.9 Å, 143.9 Å, 
> 216.3Å, 90°, 90°,  90°). The possible space group is P212121. With such a big 
> unit cell, we predicted that there are 8-10 molecules per asymmetric unit. We 
> have a decent model with sequence similarity of 49%. I tried several times 
> with Phaser search with the current model and had difficulty to find any 
> clear solution. Has anyone seen such cases and any suggestions to solve the 
> structure? Thanks!
>
> Jiyuan Ke, Ph.D.
> Research Scientist
> Van Andel Research Institute
> 333 Bostwick Ave NE
> Grand Rapids, MI 49503

Re: [ccp4bb] Crystal handling

[Ho Leung Ng]

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
 wrote:

>
> Hi all.
>
> I have small and fragile membrane protein crystals with size 40 micron. They 
> like to break when I try take them out with regular loops for freezing. Is 
> there any special tricks to handle them for data collection?
>
> Thank you.
>
> Theresa

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

[Ho Leung Ng]

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 
Subject: Who is using 64-bit Linux?

The time has come for me to upgrade my Linux OS to something more recent
for me and my student workstations. A 32-bit distro is certainly
conservative and compatible with CCP4 and Coot, but it seems like that
solution hobbles my hardware and puts some limitations on available
memory, even with PAE enabled. So who is using a 64-bit distro these
days, and are there lingering issues of compatibility and dependency
hell with commonly used XRD software, like CCP4, Coot, iMOSFLM etc.?

Ubuntu 12.04 LTS (beta) actually works OK with one simple workaround for
the global menu for CCP4 and Coot, and wine compatibility is fine for
running CrysalisPro in the same environment, so it's really comes down
to whether or not the extra performance of a 64-bit OS is worth the pain
of compatibility issues for XRD software. Any thoughts?

Cheers,

___
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: rrowl...@colgate.edu

Re: [ccp4bb] DDM

[Ho Leung Ng]

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


On Mon, Mar 26, 2012 at 1:06 PM, CCP4BB automatic digest system
 wrote:
> Date:    Mon, 26 Mar 2012 17:39:57 +
> From:    Joao Dias 
> Subject: Re: DDM
>
> Kasia,
> A lot of people uses DDM to purify membrane proteins, not a lot of people 
> crystallises them.
> If you want to crystallise a protein purified in DDM, then you should use LCP.
> If you go for vapour diffusion, you should exchange the DDM for a detergent 
> with a smaller micelle size otherwise you might get crystals but it is 
> difficult to get good diffraction. Try mixed micelles for example.
> Typically use 0.05% DDM during purification and use 100kDa cut-off membranes 
> in order to prevent detergent concentration.
> For extraction it depends on your protein and expression system but you can 
> see in the literature values between 0.5-2% being used successfully.
> Good luck.
> Cheers,
> Joao
>
> Joao Dias, Ph.D.
>
> Senior Scientist
> Heptares Therapeutics Ltd
> BioPark, Broadwater Road,
> Welwyn Garden City,
> Herts, AL7 3AX
> UK

[ccp4bb] Postdoctoral position - University of Hawaii

[Ho Leung Ng]

Applications are invited for a postdoctoral position in my lab at the
University of Hawaii at Manoa, Honolulu, Hawaii, USA. Multiple
projects are available involving receptor tyrosine kinases, GPCRs,
structure based drug design, protein engineering, and developing
methods for membrane protein crystallization. The fellow will also be
expected to come up with her own project ideas. The laboratory
benefits from collaborations with industry and the medical school to
pursue broad, interdisciplinary approaches to challenging structural
biology problems.

I am seeking a highly motivated scientist with extensive experience in
subcloning, protein expression, purification, and characterization.
Expertise with multiple expression systems including E. coli, yeast,
and cell free systems is highly valued. Experience with membrane
proteins will be very helpful. Applicants should have at least one
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

[Ho Leung Ng]

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



On Wed, Feb 22, 2012 at 5:54 PM, Bernhard Rupp (Hofkristallrat a.D.)
 wrote:
>> You might get lucky by setting up crystallization plates, but chances are
> you won't get very useful information from them, especially if your
> aggregated protein is soluble.
>
> I seem to fail to understand how crystallization plates would give
> information in the not-special case of protein aggregates NOT being soluble?
>
>
> BR
>
> Ho
>
> Ho Leung Ng
> University of Hawaii at Manoa
> Assistant Professor, Department of Chemistry h...@hawaii.edu
>

Re: [ccp4bb] Aggregated protein for crystallization

[Ho Leung Ng]

     If you haven't done so already, I would screen buffer conditions
(pH, salt concentration, glycerol, strongly reducing conditions,
ligands, detergents) by DLS to see if you can reduce aggregation. You
might get lucky by setting up crystallization plates, but chances are
you won't get very useful information from them, especially if your
aggregated protein is soluble.

     There are fluorescent dyes sensitive to aggregation state such as
ANS (anilinonaphthalene-8-sulfonate) or Nile Red. I have not used them
myself and would like to hear if others have found them useful for
screening 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!

[Ho Leung Ng]

Hello Ivan,

 You can dispense 0.1-0.2 uL drops with the PB-100 repeating
dispenser from Hamilton and a 10 uL glass syringe. But it's rather
clumsy and hard on the hands. I don't recommend using it with 96 well
plates. It's too slow to do the whole plate at one time without your
drops drying up.


Ho

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


From:xaravich ivan 
Subject: Manually setting 96 wells plates with lower volume samples!

Hi,
I apologize in advance as it is not a ccp4 related question, but over the
years, CCP4bb is synonymous with protein crystallographers virtual
university, at least for me.

Ok, now I do not have an easy access to crystallization robot, so I was
hoping if someone here have ever used the 96 well plates for manually
setting drops with much lower solution/sample volumes (0.1-0.2micro litres).

I heard about a clicker syringe that can be used to manually add lesser
volumes but I am not sure how to go about it.
Please let me know if you are aware of or  are routinely setting 96 well
trays with much lower volumes of crystallization solutions as well as
samples, manually.

thanks in advance,
ivan

Re: [ccp4bb] Protein-Protein Complex Screening

[Ho Leung Ng]

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


Date:Thu, 17 Nov 2011 14:03:44 -0600
From:Yi-Liang Liu 
Subject: Protein-Protein Complex Screening

Hi CCP4ers,

I know it is not quiet related to CCP4. I am now working on a
protein-protein complex system. I am wondering which kits I should try
in a higher priority? I appreciate everyone's suggestions, and maybe
there are some papers I can read first.

Lucas

Re: [ccp4bb] Magnetic anti-flag beads

[Ho Leung Ng]

 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
 wrote:
> From:    "Harman, Christine" 
> Subject: Magnetic anti-flag beads
>
> Hi all,
> Sorry for the off-topic question, but I wanted to find out anyone's 
> experience with using magnetic anti-flag beads (Sigma).  I have been having a 
> problem with the anti-flag antibody leaching from the beads during low pH 
> elution ( which shouldn't be happening I am thinking) and was wondering if 
> anyone has had a similar experience.  I have talked to Sigma about this and 
> they sent me another lot of beads which also appears to be leaching antibody 
> during incubation with 0.1M glycine pH 2.7 (normal elution described in 
> protocol except at pH 3.0).  I am not using the beads for purification, but 
> will be using them for protein interactions studies so sorry about the 
> off-topic question.  Given that you all are a very smart community I thought 
> I would throw this one out to you.
>
> Thanks for any brain waves on this,
>
> Christine

Re: [ccp4bb] Vivaspin vs Amicon Ultra

[Ho Leung Ng]

Hello Jan,

 I've used both with soluble and membrane proteins happily. I haven't
seen significant differences with protein behavior or performance in one or
the other, but of course, your favorite protein may differ. I've found that
buffer condition is a more important variable than membrane 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)

[Ho Leung Ng]

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

[Ho Leung Ng]

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

[Ho Leung Ng]

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?

[Ho Leung Ng]

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)

[Ho Leung Ng]

Hello Hari,

 It looks like Addgene has both pCW-LIC and pCWori available.


ho

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

[Ho Leung Ng]

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 (see getit_4), then I got the improved crystal like getit_5 after
trying seeding, different detergents, lipids and additives. But this crystal
does not diffract at all, I already tried Izit staining which shows it is
protein crystal (detergent crystal?). Does anyone have any good suggestions
for the optimization of this membrane protein crystallization? Thank you in
advance.

Best regards

Qiangmin Zhang

Biomedical Science Tower 3
Room1034
3501 Fifth Avenue
Pittsburgh, PA 15260





--
张强敏

Re: [ccp4bb] Problems in purification

[Ho Leung Ng]

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, ganesh pathare 
> wrote:
>
>> Dear all,
>>
>> I have problems in purifying a protein. The protein is 38,000 daltons and
>> has a N-ter His-Tag. The protein expression levels are low and as a result I
>> have a limit for the purification steps.
>> Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300
>> mM NaCl, 20 to 250 mM Immidazole for the affinity purification, but it
>> contains lot of impurities. I varied the salt concentrations out of which I
>> could get optimal results at 20 mM NaCl concentration but still the amount
>> of impurities was more.
>> After affinity purifications I used Ion exchange chromatography using MonoQ
>> column (25 mM tris pH 7.5,  NaCl 0 to 1M) which could not seperate the
>> protein from the impurities. I also tried using Hydrophobic interaction
>> chromatography (Resource Ether, Phenyl sepharose, Resource
>> Isopropyl) instead of ionexchange chromatography, which resulted in
>> better purification of the protein, but the problem is I get very less
>> protein after this step and there are still two major impurities. The buffer
>> conditions for HIC was (1.5 M ammonium sulphate, 25 mM Phosphate buffer pH
>> 7).
>>
>>
>> I would be very greatful if someone could help me in this concern.
>> Thanks in advance.
>>
>> Regards,
>> Ganesh
>>

Re: [ccp4bb] Crystallizing a membrane associated protein

[Ho Leung Ng]

Hi Dan,

 I agree with Bert's general approach. How does your purified
protein look under size exclusion chromatography or DLS? Do you have a
functional assay?


ho

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

[Ho Leung Ng]

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 of the protein in detergent.


ho

Re: [ccp4bb] Beginning crystallography text

[Ho Leung Ng]

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

[Ho Leung Ng]

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.

You may want to read Gill, "Evaluating the efficacy of tryptophan
fluorescence and absorbance as a selection tool for identifying
protein crystals", Acta Cryst F 66:364, which compares several
microscopes.
http://www.ncbi.nlm.nih.gov/pubmed/20208182


ho



Hi there.

I don't know how much it costs, but I've used a Korima
PRS-1000 serval
times, and it appears to be fairly good. Although the image quality isn't
great and there is still a bit of a learning curve for identifying small
crystals and/or crystals buried in precipitate, I've found this microscope
to be a very valuable tool.

-Joel

=
Joel M. Guenther
PhD Candidate, Department of Chemistry
Kuriyan Laboratory
http://jkweb.berkeley.edu/
University of California, Berkeley
176 Stanley Hall, QB3
Berkeley, CA 94720-3220
=

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

[Ho Leung Ng]

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:"Soisson, Stephen M" 
Subject: Re: Converting cif-files into pdb-files / reading cif -files into coot

There is a time-honored tradition of PhD students struggling through
format conversion issues.  Where is the fun and "learning opportunity"
if everything ran smoothly? ;)

Cheers,

Steve

Re: [ccp4bb] please recommend a crystallization incubator

[Ho Leung Ng]

I've been quite happy with our refrigerated BOD incubator from Fisher.
Relatively inexpensive.



ho

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

[Ho Leung Ng]

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


ho

Re: [ccp4bb] Cryo Vs crystal size

[Ho Leung Ng]

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

[Ho Leung Ng]

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

[Ho Leung Ng]

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 
Subject: activation of thiol group

Dear colleagues:
We have a 1.4 Angstrom structure of an enzyme showing a water molecule
enclosed in a triangular pocket formed by the hydroxyl oxygens of 2 serine
residues and a sulfhydryl group of an essential cysteine. The water molecule
is forming a 2.8 Angstrom hydrogen bond with each of the hydroxyl groups of
the 2 serines and a 2.9 Angstrom hydrogen bond with the sulfhydryl group of
the cysteine. Is it possible for such a water molecule to lower the pKa of
the cysteine and activate the thiol group?
I would appreciate any comments or suggestions or information on any
literature that I need to look up :>
Sincerely,
Deepak

Re: [ccp4bb] TCEP effect on protein

[Ho Leung Ng]

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
 wrote:
> There are 5 messages totaling 490 lines in this issue.
>
> Topics of the day:
>
>  1. TCEP effect on protein (4)
>  2. 3 PhD studentships available immediately
>
> --
>
> Date:    Thu, 25 Mar 2010 22:51:30 -0800
> From:    megha goyal 
> Subject: TCEP effect on protein
>
> Hi all,
>
> My protein is relatively pure except the dimer. i used 10 mM TCEP to reduce
> it (directly added 1 M TCEP to make final volume of 10mM and kept at room
> temperature for 10 mins] then i dialysed the protein sample to remove TCEP
> [dialysis buffer is Na acetate pH 4.0].
>
> On performing SDS PAGE analysis after dialysis of the protein we are getting
> no dimer band, only band of our protein is observed and same is the case
> with UV reading there is no change in it. But the HPLC analysis of the
> protein shows two peaks instead of one peak as observed before TCEP
> treatment. what can be the reason for this. Kindly guide. i need the protein
> to formulate and conduct stability studies on the sample. the protein we
> obtain after IEX is pure except the dimer and i do not want to go for SEC as
> it greatly reduces protein content and also is quite time consuming. any
> light on what is happening will bevery useful.
>
> thanks and regards
>
> --
>
> Date:    Fri, 26 Mar 2010 08:37:43 +0100
> From:    Ganesh Natrajan 
> Subject: Re: TCEP effect on protein
>
>
>
> Hi Megha,
>
> The two peaks on the HPLC indicate that your protein is
> existing in a monomer-dimer equilibrium in solution. The dimerisation is
> most probably caused by disulphide bridges. The use of TCEP is breaking
> those disulphides and that is causing the equilibrium to move towards the
> monomeric state. However, when the TCEP is dialysed out, the disulphides
> start forming again and this is causing the equilibrum to move towards the
> dimeric state, a process clearly hastened by the strongly oxidising pH 4 of
> the dialysis buffer.
>
> Now it all depends on what you want to do. If you
> want to use the protein in a (largely) monomeric form, I would recommend
> that you don't dialyse out the TCEP.
>
> regards
>
> Ganesh
>
>
> **
> Blow, blow, thou winter
> wind
> Thou art not so unkind
> As man's ingratitude;
> Thy tooth is not so
> keen,
> Because thou art not seen,
> Although thy breath be rude.
>
> -William
> Shakespeare
> **
>
> On Thu, 25 Mar
> 2010 22:51:30 -0800, megha goyal  wrote:  Hi all,   My protein is
> relatively pure except the dimer. i used 10 mM TCEP to reduce it (directly
> added 1 M TCEP to make final volume of 10mM and kept at room temperature
> for 10 mins] then i dialysed the protein sample to remove TCEP [dialysis
> buffer is Na acetate pH 4.0].   On performing SDS PAGE analysis after
> dialysis of the protein we are getting no dimer band, only band of our
> protein is observed and same is the case with UV reading there is no change
> in it. But the HPLC analysis of the protein shows two peaks instead of one
> peak as observed before TCEP treatment. what can be the reason for this.
> Kindly guide. i need the protein to formulate and conduct stability studies
> on the sample. the protein we obtain after IEX is pure except the dimer and
> i do not want to go for SEC as it greatly reduces protein content and also
> is quite time consuming. any light on what is happening will bevery useful.
>  thanks and regards
>
> --
>
> --
>
> Date:    Fri, 26 Mar 2010 08:49:55 +0100
> From:    Matthias Zebisch 
> Subject: Re: TCEP effect on protein
>
> Hi Ganesh and Mega!
>
> I do not agree with Ganesh. I assume, Megha, that truly reversed
> phaseHPLC was used. This is a denaturing method and the natural
> disulfide should not form again during the run.
> Also pH 4 can not be described "oxidizing". Actually, reduced proteins
> are often dialyzed against acidic buffers to prevent disulfide formation
> via the thiolate anion.
> Still, a reducing agent may be used during the run?
>
> Sorry that I can not offer a solution to the real problem. More
> experimental details may be necessary.
>
> Bets regards, Matthias
>
> Am 3/26/2010 8:37 AM, schrieb Ganesh Natrajan:
>>
>> Hi Megha,
>>
>> The two peaks on the HPLC indicate that your protein is existing in a
>> monomer-dimer equilibrium in solution. The dimerisation is most
>> probably caused by disulphide bridges. The use of TCEP is breaking
>> those disulphides and that is causing the equilibrium to move towards
>> the monomeric state. However, when the TCEP is dialysed out, the
>> disulphides start forming again and this is causing the equilibrum to
>> move

Re: [ccp4bb] how to improve resolution

[Ho Leung Ng]

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 the reservoir (see Janet Newman, Acta Cryst
D, 61:490).


ho


From:rui 
Subject: how to improve resolution

--0016e6d7ee96984676047ed308d5
Content-Type: text/plain; charset=ISO-8859-1

Hi, All,

We are trying to crystallize a protein and found some initial hit in the
following conditions,

pH 4.8, 0.2 M AS or some other salts ( NaCl,LiCl, MgCl2 ), 32% PEG4000 or
PEG3350 ). However the quality of the crystal is not so great,some of them
look like needle cluster(very long in length), some of them look like
multi-crystals or hollow inside. We tried to optimize the pH and PEG and
tested one that diffracts at 2.9A. For the next, how to improve
resolution?Any suggestions? Even mutate the protein to get a high resolution
is ok, generally what kind of mutation would make proteins crystallize
better? Thanks.

Re: [ccp4bb] Crystal rescue

[Ho Leung Ng]

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

[Ho Leung Ng]

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
ConfometRx


Date:Mon, 25 Jan 2010 18:49:14 +0800
From:"Dr. STEPHEN SIN-YIN, CHUI" 
Subject: Purchase of Protein Samples

Dear all CCP4 experts,

Hello, I'm not molecular biologist and quite new in this field. I would like to
know what companies (i.e. Sigma/Aldrich) are possibe for me to purchase
purified protein samples. Any suggestion is welcomed.

Many thanks in advance.

stephen

--
Dr. Stephen Sin-Yin Chui
Research Assistant Professor,
Department of Chemistry,
The University of Hong Kong, Pokfulam Road,
Hong Kong SAR, China.
Tel: 22415814 (Office), 22415818 (X-ray Diffraction Laboratory)

Re: [ccp4bb] smeared spot in diffraction

[Ho Leung Ng]

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 cryoprotect., etc. If possible, try to grow
the crystals in a condition that can be directly flash frozen.

2. This isn't often brought up, but I've frequently seen sharper
diffraction from smaller crystals due to better freezing/fewer crystal
defects.


Good luck!
ho

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

[Ho Leung Ng]

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 techniques to improve expression. What has worked best
for me in the past were 1) lowering the temperature of
induction/expression and 2) using different tags.

 It sounds like you've already tried many different parameters.
Consider different expression hosts.


ho

Re: [ccp4bb] contaminants in PEG - references

[Ho Leung Ng]

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...

[Ho Leung Ng]

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

[Ho Leung Ng]

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
http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-477.html


ho
Confometrx

Re: [ccp4bb] Reducing Agent Tips?

[Ho Leung Ng]

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

[Ho Leung Ng]

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

[Ho Leung Ng]

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

[Ho-Leung Ng]

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
(nihserver.mbi.ucla.edu/SER/) to predict which lysines are the best
targets for mutagenesis.


ho
UC Berkeley

Re: [ccp4bb] MAD phasing

[Ho-Leung Ng]

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?

[Ho-Leung Ng]

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

--
Date:Mon, 18 May 2009 09:34:12 -0400
From:Annie Hassell 
Subject: Re: How to improve crystal which is twinning?

Heng--

Two things you might want to try:

1.  Drop ratios with your current conditions and DMSO additive.  Use=20
different concentrations of DMSO too.=20

2.  Try using Hampton Crystal Screen and Crystal Screen 2 as additives.  I =

generally do this by adding 5% of XS 1 & 2 to the wells.  Once I find one=20
of these  reagents that  improves crystal quality/diffraction, I optimize=20
the concentration for that.  You can do this with the Mosquito robot for=20
96 well formator in the 24 well VDX trays.=20

Hope this helps!
annie




Annie  Hassell
Glaxo Smithkline
5 Moore Drive
RTP, NC  27709
919/483-3228
919/483-0368 (FAX)
annie.m.hass...@gsk.com

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

[Ho-Leung Ng]

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 protein:crystallization solution ratios carefully.


Good luck!
Ho
UC Berkeley

[ccp4bb] Glycerol as metal chelator?

[Ho-Leung Ng]

 I am working with a metalloprotein that binds cobalt and iron. I
was surprised that the solved structures showed the crystals
cryoprotected with glycerol are metal free while crystals
cryoprotected with ethylene glycol had the metals present. Both
cryoprotectant solutions contained metal in the 10 mM range and are
buffered at pH 9. I assume glycerol must be a weak chelator otherwise
it wouldn't be so ubiquitous in protein biochemistry. Has anyone else
experienced this before with glycerol?


Ho
UC Berkeley

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

[Ho-Leung Ng]

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_zbySqumaA&feature=related

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

[Ho-Leung Ng]

Hi Bryan,

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


ho

Re: [ccp4bb] Lost my protein

[Ho-Leung Ng]

Is your protein sticking to the membrane or crashing/aggregating from
the concentrating?

Try to find a buffer condition in which your protein is more soluble
and less aggregation prone. You can try varying salt concentration,
pH, and inclusion of additives like glycerol or detergent.


Ho
UC Berkeley

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

[Ho-Leung Ng]

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 strain of E. coli. Whether it is or isn't an expression host
depends on the definition, and I am not going to argue semantics.

The T5 promoter works with regular garden variety RNApol of E. coli.
Therefore ANY E. coli strain is an 'expression host' for vectors that
contain this promoter.

I've expressed many proteins in XL1-Blue and I see no reason why you can't
express yours, either.

Artem

Re: [ccp4bb] Cryo-protectant

[Ho-Leung Ng]

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 PEG in your reservoir to 40% and allow vapor diffusion
to gently dehydrate your crystals to allow direct freezing.


ho
UC Berkeley

Re: [ccp4bb] protein purity in SAXS

[Ho-Leung Ng]

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

[Ho-Leung Ng]

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] soaking with Samarium chloride

[Ho-Leung Ng]

Hello Amit,

 Some great resources for general information are:

http://eagle.mmid.med.ualberta.ca/tutorials/HA/
Agniswamy et al, Acta Cryst D 64, 354
http://www.doe-mbi.ucla.edu/~sawaya/tutorials/Phasing/references.html

Recent threads also discussed halide soaks and Artem Evdokimov's
protocol for iodinating tyrosines.

We've also had success mutating residues to methionine followed by
SeMet phasing. I'd do this if the easy stuff doesn't work.


Ho
UC Berkeley

--

Date:Mon, 6 Apr 2009 20:16:18 +0100
From:amit sharma <3112a...@gmail.com>
Subject: soaking with Samarium chloride

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Dear All,

I have a crystal growing in the presence of 0.1M Sodium Acetate pH 5.0, 10%
PEG4000, 7.5% Dioxane and 10% Ethylene Glycol. I wanted to know if it would
be alright to use Samarium chloride for derivatization. I am worried about
the leaching of samarium by acetate. Also, what soak times can I use and
what conc. of Samarium chloride? My protein is 10 kDa , has no
cysteines/methionines and  pI for the molecule is 4.6. Also, could somebody
suggest what other heavy atom soaks could be performed in this case?

Any suggestions would be appreciated.
Many thanks in advance

--
Amit Sharma

Re: [ccp4bb] Halide soaking

[Ho-Leung Ng]

 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 haven't had
any success with those yet.


ho
UC Berkeley

Re: [ccp4bb] Design Constructs

[Ho-Leung Ng]

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. However, as Artem noted, the results can be
unpredictable, especially with regard to expression. You may have to
go with a brute force approach. For a case like yours, I might try
deleting every 3-4 residues, which only leaves you half a dozen or so
constructs to test. Definitely test both sequence clusters you
describe.


Good luck!
ho
UC Berkeley


-
Date:Mon, 30 Mar 2009 14:11:02 +0100
From:Haridasan Namboodiri 
Subject: Design Constructs

Hello

I am designing a protein construct for structural biology. It is a protei=
n kinase=20
which has not been crystallized earlier. I was comparing the kinase domai=
ns of=20
other closely related family members characterized biochemically vs=20
crystallization constructs. For crystallography constructs, there are dif=
ferent=20
stretches of amino acid residues particularly at the N-terminus (some con=
tain=20
extra 2-5 residues while others have 15-20 residues.

My question:  Is  there a rational way of designing exact constructs one=20=

would propose to make, eg., by a sequence alignment showing nearest=20
homology neighbors that guided construct design etc..


Sincerely
Hari

Haridasan V. M. Namboodiri, PhD
Scientist-Structural Biology
Locus Pharmaceuticals, Inc.
Four Valley Square
512 Township Line Road
Blue Bell, PA 19422
email: vnambood...@locuspharma.com
Ph:  215-358-2012
Fax: 215-358-2020

Re: [ccp4bb] selenomethionine labeling

[Ho-Leung Ng]

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 
Subject: selenomethionine labeling

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Dear All:

 I got a problem with the expression of one N-terminal His6 tagged pro=
tein in B834 cells when I tried to do selenomethionine labeling.=20

  However=2C the protein can be expressed in BL21{DE3) cells.=20

  Welcome any troubleshooting tips.

  All the best=2C

Jerry

Re: [ccp4bb] Problems with phasing a protein (1300aa)

[Ho-Leung Ng]

I think you need to first address the issue of radiation damage.
Radiation damage can prevent you from solving the structure even
before it affects Rsym or I/sigma.

I haven't merged data from multiple crystals to solve a MAD structure,
but I'd try it in your case, with inverse beam data collection. You
will have to be conservative with how many frames you keep per
crystal, tossing out frames well before you see the obvious effects of
radiation damage.

I've heard of people using ascorbic acid to radioprotect their crystals.

Were your native crystals also so radiation sensitive? If so, you
could also try mutating away cysteines, which are often the most
sensitive.


Ho
UC Berkeley


From:Kumar 
Subject: Problems with phasing a protein (1300aa)

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Hello CCP4bb members,

I have been trying to obtain phases for a protein which contain ~1300aa. We
have obtained native data to a resolution of 3.3A (Space group I222 or
I212121). But we are having tough time phasing it.

'Se' labeled crystals diffracts maximally up to 3.5 to 4 A and dies very
quickly on most of the beamlines. We have scanned at Se wavelength and it
gives very strong signal as it contain ~45 Se in AU (1300 aa). It is
difficult to collect a complete dataset (maximally we get 50-60 % completion
with Rmerge ~15) out of one crystal on regular beamline. At microfocus
beamline (APS), we were able to collect data in 3-4 batches and merge them
to get a complete dataset (Rmerge ~18-20) out of one crystal. We used data
collected on microfocus beamline (at peak wavelength) for locating heavy
atom position using SHELXD, Solve and Phenix.hyss. SOlve and Phenix.hyss
find very few heavy atom sites 1-5 whereas SHELX-CDE lists many but shows no
difference in original and inverted (contrast and connectivity). Our phasing
attempts with datasets obtained after merging two incomplete dataset from
two different crystal has also been disappointing.

My another worry is absolute value of average intensity, which seems to be
quite low in most of the datasets. Below I have pasted last table of
scale.log (HKL2000).
Shell Lower Upper Average  Average Norm. Linear Square
limitAngstrom   I   error   stat. Chi**2  R-fac  R-fac
 50.00   7.5345.4 1.6 1.3  1.295  0.055  0.047
 7.53   5.9811.4 1.3 1.3  0.672  0.135  0.114
  5.98   5.2311.2 1.6 1.6  0.643  0.171  0.152
 5.23   4.7516.8 2.0 1.9  0.736  0.148  0.118
  4.75   4.4118.8 2.2 2.2  0.739  0.143  0.132
 4.41   4.1514.6 2.4 2.4  0.653  0.190  0.175
  4.15   3.9411.3 2.5 2.5  0.582  0.247  0.226
 3.94   3.7710.1 2.8 2.8  0.511  0.280  0.191
  3.77   3.63 8.0 3.1 3.1  0.450  0.315  0.285
 3.63   3.50 7.6 3.3 3.2  0.483  0.311  0.270
 All reflections 15.5 2.3 2.2  0.694  0.153  0.106

Now, I want you to help me by answering some of my queries:

1. Is it possible to get MAD/SAD phasing done from a dataset having more
than 15% Rmerge and resolution in the range of 4 - 4.5 Ang?

2. Will a complete data set obtained from merging various batches(30-40
frames each) from one or more than one crystal will have proper anomalous
signal for phasing? I am worried as weak anomalous signal may get lost while
merging.

3. Will such a low value of average Intensities (as shown above from HKL
scale log file) will be good enough for MAD/SAD phasing or I really need to
improve crystal quality for stronger diffraction.

4. For MAD/SAD phasing, till what resolution we need to have anomalous
signal ? Many of my datasets shows anomalous signal maximally up to 6-8 A
(calculated using Phenix.xtriage).

5. Since I have low resolution (3.5 to 4 A)data, relatively high Rmerge
(14-15%), lower value of average intensity, anomalous signal up to 6 A or
so. which programs will be more useful for heavy atom location and to
prevent false positives from being selected?

We have been also trying our luck with heavy atom soak but that also has not
been very encouraging. I would appreciate any suggestions in this regard.
Thanks in advance and sorry for such a long mail.
Kumar

Re: [ccp4bb] Purification

[Ho-Leung Ng]

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 
> Subject: purification
>
> Hello everyone,
>
> I am expressing a 100 KDa eukaryotic membrane protein in E coli. The prot=
> ein
> is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KD=
> a.
>
> However, the protein get severely degraded so after putting through a Ni-=
> NTA
> column, the protein came out with a lot of contaminant bands. I did a
> western blot using antibody against his tag. The total cell lysate gave
> signals in many bands. The flow through did not give any signal and the
> eluted fraction again gave many band signals, indicating the protein got
> degraded copiously even before purification.
>
> I used Roche protease inhibitor tablet and still got a lot of degradation=
> .
> Can anyone suggest a way to avoid the problem or a purification method so=
>
> that I can purify the intact protein while keeping away the unwanted
> degraded fractions.
>
> Thanks heaps in advance.
>
> Kien

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

[Ho-Leung Ng]

 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] CCP4BB Digest - 11 Mar 2009 to 12 Mar 2009 (#2009-71)

[Ho-Leung Ng]

Hampton sells a Silver Bullets screen that may work for you.


ho
UC Berkeley
--
Date:Thu, 12 Mar 2009 12:15:18 -0700
From:Dinesh Palanivelu 
Subject: Fragment Screening kit

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Dear all,
We have been trying to crystalize a membrane protein in which the
function is unknown. We got some initial crystal hits and we are in
the process of optimization.
I am looking for a screening kit for identifying the potential ligand/
substrates (small molecule metabolites) of an unknown target by co-
crystallization experiments.

Any suggestions will be appreciated.
Thanks in advance.

Cheers
Dinesh

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

[Ho-Leung Ng]

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 
Subject: Re: Off topic: Mammalian gene expression in E. coli

 Just in case anyone else encounters the same problem, like Artem I=20
have had a few SUMO fusion constructs that have stubbornly refused to=20
cleave even with a 1mg of enzyme (Ratio ~ 1:100) at 37c in the presence=20
of low concentrations of chaotrope. In all cases the problem was solved=20
by inserting a glycine residue between the cleavage site and the first=20
amino acid (always a methionine in my case). This resulted in same=20
constructs being able to be cleaved at 4oC, with a Ratio 1:1000 to=20
1:2000 of hydrolase in 30 minutes (Cleavage was performed in a tyipcal=20
IMAC elution buffer with 250 mM NaCl). Of course by doing this you will=20
no longer have the "Native" amino terminus on you protein but funnily=20
enough have the same additional residue that TEV leaves behind. I=20
perform all my cloning using LIC but if I remember correctly the NYSGC=20
use a BamHI (GGATTC) for cloning downstream of the SUMO cleavage site=20
which will introduce an additional serine residue. I assume, without=20
ever seeing their cleavage data, that they never had a cleavage problem.

In the one case where I finally got crystals of the protein that=20
initially poorly cleaved as a SUMO fusion construct the amino terminus=20
was highly ordered in the structure (I could use the Selenium labeled=20
start methionine for phasing). I'm curious if this can be extrapolated=20
to all poorly cleavable fusion constructs.

Stephen

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

[Ho-Leung Ng]

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. Also try changing from an N-terminal to
C-terminal tag (or vice-versa).


ho
UC Berkeley

[ccp4bb] include or exclude overloads

[Ho-Leung Ng]

Hi Clemens,

 Thank you for the clarification. I had thought you were
advocating using a general low resolution cutoff, with which I would
disagree. I spend a lot of time troubleshooting data collected and
processed by other people. Those are good reminders to go back and
check beamstop settings and overloaded spots.


Phil,

 I was thinking specifically of what to do with overloads when a
short exposure pass wasn't done, when I asked what should be done with
poorly measured data. Exclusion gives you zeroes instead of what
should be the highest intensity spots in your data set. But inclusion
could throw off scaling, which would be worse. SCALA by default
rejects estimated intensities from overloads from mosflm, which I
presume is for a very good reason. Would it be better to not use the
estimated intensities for scale determination but keep them in the
data set?


ho
UC Berkeley

> --
>
> Date:Mon, 16 Feb 2009 09:07:38 +
> From:Clemens Vonrhein 
> Subject: Re: CCP4BB Digest - 12 Feb 2009 to 13 Feb 2009 (#2009-45)
>
> Dear Ho,
>
> On Fri, Feb 13, 2009 at 04:45:29PM -0800, Ho-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 completely agree: it also "bothers me to discard data". However, the
> crucial word here is 'data' - which is different from Miller indices
> HKL.
>
> So I am mainly concerned with two types of reflections (HKL) that
> aren't really 'data':
>
>  1) overloads
>
> These are obviously not included into your final reflection file
> (unless you explicitely tell the integration software to do that
> - in which case you know exactly what you are doing anyway). So
> there is no problem ... or is there?
>
> Overloaded reflections are only very few at low resolution - and
> the most important reflections are obviously the ones at 1.94A
> resolution so that one can have a 'better-than-2A' structure in
> the end ... ;-) ... So still no problem, right?
>
> And who cares if the completelness of the data isn't 100% but
> rather 99.4%? Exactly ... so where is the problem?
>
> But: these few missing reflections are systematically the
> strongest ones at low(ish) resolution, and any systematically
> missing data is not a good thing to have.
>
> Solution: always collect a low-intensity pass to measure those
> strong reflections if there is a substantial amount of overloads.
>
>  2) beamstop
>
> The integration software will predict all reflections based on
> your parameters (apart from the 000 reflection): it doesn't care
> if such a reflection would be behind the beamstop shadow or
> not. However, a reflection behind the beamstop will obviously not
> actually be there - and the integrated intensity (probably a very
> low value) will be wrong.
>
> One example of such effects in the context of experimental
> phasing is bogus anomalous differences. Imagine that your
> beamstop is not exactly centred around the direct beam. You will
> have it extending a little bit more to one side (giving you
> maybe 20A low resolution) than to the other side (maybe 30A
> resolution). In one orientation of the crystal you might be able
> to collect a 25A (h,k,l) reflection very well (because it is on
> the side where the beamstop only starts at 30A) - but the
> (-h,-k,-l) relfection is collected in an orientation where it is
> on the 20A-side of the beamstop, i.e. it is predicted within the
> beamstop shadow.
>
> Effect: you have a valid I+ measurement but a more-or-less zero
> I- measurement, giving you a huge anomalous difference that
> shouldn't really be there.
>
> Now if you measured your data in different orientations (kappa
> goniostat) with high enough multiplicity, this one bogus
> measurement will probably be thrown out during
> scaling/merging. You can e.g. check the so-called ROGUES file
> produced by SCALA. But if you have the usual multiplicity of only
> 3-4 the scaling/merging process might not detect this as an
> outlier correctly and it ends up in your data. Sure, programs
> like autoSHARP will check for these outliers and try to reject
> them - but this is only a hack/fix for the fundamental problem:
> telling the integration program what the good area of the
> detector is.
>

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

[Ho-Leung Ng]

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 Berkeley

> Date:Fri, 13 Feb 2009 17:14:38 +
> From:Clemens Vonrhein 
> Subject: Re: unstable refinement

> * resolution limits: are you suddenly including all those poorly
>  measured or non existent reflections at the low resolution end (10A
>  and lower) that are only present because the beamstop masking wasn't
>  don properly during data processing/integration?
>
>  These bogus reflections can mess up your bulk-solvent correction and
>  scaling with weird effects. Better check the scale factors you're
>  getting during refinement and if they make sense.
>
>
> Cheers
>
> Clemens
>
> ***
> * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
> *
> *  Global Phasing Ltd.
> *  Sheraton House, Castle Park
> *  Cambridge CB3 0AX, UK
> *--
> * BUSTER Development Group  (http://www.globalphasing.com)

Re: [ccp4bb] 2D

[Ho-Leung Ng]

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

-
It mostly means little intermolecular contacts in one direction because=20
of charge repulsion, shape incomplementarity etc etc.

One thing to try is screen for additives that can help to make more=20
contacts between the layers or,  screen for new crystal forms using=20
microseed matrix screening!

- J. -

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

[Ho-Leung Ng]

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 maps useful in combating phase bias. I suggest you try to get
experimental phases. It can save you a lot of time struggling with a
difficult MR problem and rebuilding into lousy maps.

 I'd love to know how you end up solving the structure!


ho
UC Berkeley

Re: [ccp4bb] MR with DNA

[Ho-Leung Ng]

It would be difficult because your protein probably affects the DNA
conformation. Do you have some idea how your protein affects DNA
conformation? But I think a brute force strategy trying every piece of
DNA in the PDB as a template might work. Another approach would be to
do MR using only segments of your DNA that might be straight B-DNA.


ho

-
Date:Fri, 5 Dec 2008 16:32:14 -0600
From:UT MDACC <[EMAIL PROTECTED]>
Subject: MR with DNA

Is there any existing example about anybody trying to do molecular
replacement for a protein-DNA complex using any B-DNA from the pdb database.
I am wondering 1) whether it is doable 2)if it is not doable, why not?
Thanks in advance for the feedback

[ccp4bb] SUMMARY - crystallization of proteins with His-tag and/or c-myc tags

[Ho-Leung Ng]

An example of using MBP as a crystallization tag:

Ke A, Wolberger C. Insights into binding cooperativity of
MATa1/MATalpha2 from the crystal structure of a MATa1
homeodomain-maltose binding protein chimera.
Protein Sci. 2003 Feb;12(2):306-12


Ho

Re: [ccp4bb] CCP4BB Digest - 3 May 2007 to 4 May 2007 (#2007-47)

[Ho-Leung Ng]

Yes, as an example, you can look at Acta Cryst. (2001). D57, 213-218


ho


Date:Fri, 4 May 2007 14:40:07 -0700
From:Shane Atwell <[EMAIL PROTECTED]>
Subject: Nucleotide duplexes by direct methods

A friend of mine asked me whether small nucleotide duplexes can be
solved by direct methods. I think their data is 2.0A.