Re: [ccp4bb] Phasing with Many Monomers/AU

[Felix Frolow]

Francis, It can happened
We have (not yet published)  P1 with 24 molecules. When we cut His-tag we get 
P1 with 32 molecules. 
In our case we believe it is dictated by very strong interaction between two 
monomers, and strong interaction between dimers with build a flattish tetramer. 
Probably such formations 
is more difficult to oaks than globular oligomers. 
In this moment I do not recall what we see in solution, I have to check.
Relating to structure solution, P1 is very convenient space group.
I would go for determination this structure by SAD (SHELXC/D/E pipe, PHENIX or 
SHARP). For the native - molecular replacement.
In our time after tremendous developments in Refmac and Phenix and development 
o DM refinement is 3-3.4 Ang. Is not very difficult.
I would use in addition to NCS restraints in refinement also multi crystal 
averaging. Roumors say it is the most strongest phasing method (attributed to 
Eleanor Dodson, myself never used it).


FF

Dr Felix Frolow   
Professor of Structural Biology and Biotechnology, Department of Molecular 
Microbiology and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On Jan 19, 2014, at 08:48 , Francis Reyes  wrote:

> You sure about this space group? 24 monomers in P1 is unusual (at least to me)
> 
> F
> 
>> On Jan 18, 2014, at 9:14 AM, Chris Fage  wrote:
>> 
>> Hello Everyone,
>> 
>> I am currently trying to phase a structure with an asymmetric unit predicted 
>> to contain 20-24 monomers (space group P1). The native crystals, while 
>> beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms 
>> at best, and SeMet-derived crystals grow with poor morphology (small 
>> needles). Also, based a fluorescence scan, I know that mercury does not bind 
>> appreciably. Other than screening for a new space group, what options might 
>> I have for phasing this many monomers at lower resolution? Is there any real 
>> chance of solving the structure in this space group?
>> 
>> Thank you in advance for any suggestions!
>> 
>> Regards,
>> Chris
>> 

Re: [ccp4bb] Phasing with Many Monomers/AU

[Francis Reyes]

You sure about this space group? 24 monomers in P1 is unusual (at least to me)

F

> On Jan 18, 2014, at 9:14 AM, Chris Fage  wrote:
> 
> Hello Everyone,
> 
> I am currently trying to phase a structure with an asymmetric unit predicted 
> to contain 20-24 monomers (space group P1). The native crystals, while 
> beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms 
> at best, and SeMet-derived crystals grow with poor morphology (small 
> needles). Also, based a fluorescence scan, I know that mercury does not bind 
> appreciably. Other than screening for a new space group, what options might I 
> have for phasing this many monomers at lower resolution? Is there any real 
> chance of solving the structure in this space group?
> 
> Thank you in advance for any suggestions!
> 
> Regards,
> Chris
> 

Re: [ccp4bb] Phasing with Many Monomers/AU

[Scott Thomas Walsh]

Hi Chris,

It would be nice to have a wee bit more information.  Is 3.4-3.0 angstroms from 
a home source
or synchrotron?  What are the crystallization conditions for both the native 
and SeMet crystals?
Did you see the SeMet crystals with the native crystals.  Have you tried MMS 
with the native crystals
into new screening conditions.

Cheers,

Scott

On Jan 18, 2014, at 12:14 PM, Chris Fage  wrote:

> Hello Everyone,
> 
> I am currently trying to phase a structure with an asymmetric unit predicted 
> to contain 20-24 monomers (space group P1). The native crystals, while 
> beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms 
> at best, and SeMet-derived crystals grow with poor morphology (small 
> needles). Also, based a fluorescence scan, I know that mercury does not bind 
> appreciably. Other than screening for a new space group, what options might I 
> have for phasing this many monomers at lower resolution? Is there any real 
> chance of solving the structure in this space group?
> 
> Thank you in advance for any suggestions!
> 
> Regards,
> Chris
> 

[ccp4bb] Cryo-EM postdoctoral position at University of Auckland

[Alok Mitra]

Dear Colleagues:

Posdoctoral position at the School of Biological Sciences University of Auckland
We are seeking a well-qualified and highly motivated candidate for the position 
of a postdoctoral Research Fellow to carry out structure/function studies of 
anti-feeding prophage (Afp) using high-resolution cryo-EM. The project in 
collaboration with Dr. Mark Hurst of Ag Research is part of a 
multi-disciplinary study supported by the Royal Society of New Zealand Marsden 
Fund.
Afp is a novel bacteriophage tail-like particle that is pathogenic to New 
Zealand grass grub (1) Hurst et al. (2004) J. Bacteriol. 186:5116-5128; 2) 
Hurst et al. (2007) FEMS Microbiol. Lett. 270:42-48) through the delivery of a 
protein toxin. This microinjection device, formally included in the type VI 
secretion system family, is composed of eighteen different gene products and we 
are revealing the roles of the different constituting proteins in the assembly 
of Afp and its mechanism of action by a combination of high-resolution cryo-EM 
and molecular biology approaches (1) Heymann, J. B., Bartho, J. D., Rybakova, 
D. et al. (2013) 3-dimensional structure of the toxin-delivery particle 
antifeeding prophage of Serratia entomophila. J. Biol. Chem. 288: 25276-25284. 
2) Rybakova, D., Radjainia, M., et al. (2013). Role of antifeeding prophage 
(Afp) protein Afp16 in terminating the length of the Afp tailocin and 
stabilizing its sheath. Mol. Microbiol. 89: 702-714.).
The selected candidate preferably should have prior experience in Structural 
Biology as that involved in high-resolution cryo-electron microscopy and image 
processing. Familiarity with recombinant protein expression and/or X-ray 
crystallography will be a plus and he/she will interact vigorously with the 
Hurst Lab.
The cryo-EM facility at UoA is the premier such facility at New Zealand. We 
have a FEI T120 (cryo) and CM12 for routine experiments and a TF20 equipped 
with GIF for high-resolution cryo-EM and cryo-tomography work. Other ancillary 
equipment include a FEI vitrobot, 2 manual plunge-freeze devices, a plasma 
cleaner, several GATAN 626 (60° and 70°) and one 914 (80°) cryo-stage. The 
computing facility within our laboratory and the access to computing cluster at 
UoA is sufficient for large-volume image processing. We also have access to an 
International class X-ray crystallography facility in house.
University of Auckland has a vibrant research environment and is the premier 
such Institute in New Zealand. Auckland is a multi-ethnic cosmopolitan city 
consistently ranked amongst the top ten cities in the world. New Zealand is 
well known for its scenic beauty.
For further information, please send a current CV and contact Dr. Alok K. 
Mitra, School of Biological Sciences, University of Auckland, Auckland, New 
Zealand. Tel: +64 09 923 8162, E.mail: 
a.mi...@auckland.ac.nz

[ccp4bb] Postdoctoral Fellow in Structural Virology

[Ke]

Postdoctoral Fellow in Structural Virology

Institute for Molecular Virology

University of Minnesota‐Twin Cities

Minneapolis, MN 55455

Web: www.virology.umn.edu


 Description

A Postdoctoral Fellow is being sought for a collaboration involving the
application of cryo‐electron tomography  (cryo‐ET) of human T‐cell leukemia
virus particles. This is a collaborative research project between
the laboratories of Profs. Wei Zhang, Joachim Mueller and Louis Mansky.
 Scientists recruited will join a strongly interactive, collaborative and
collegial research environment. Minneapolis is a dynamic and progressive
city with outstanding cultural attractions and a high standard of living.


 Skills

Candidates are expected to have a strong background in (cryo‐)electron
microscopy /tomography/image processing and analysis or other structural
biology techniques such as X‐ray crystallography or NMR spectroscopy.
Experience in molecular and cell biology/biochemistry/molecular virology is
a plus.


 Resources

The cryo‐ET data collection will be performed using the TF30 Tecnai FEG
TEM, which is housed in the Characterization Facility (
http://www.charfac.umn.edu). The microscope includes a Gatan image filter
for electron energy loss spectroscopy and energy filtered imaging, as well
as high angle annular dark field detector for Z‐contrast imaging.
 Xplore3D, SerialEM, UCSF tomo and Amira software are available for
TEM/STEM Tomography.  The facility is also equipped with a FEI vitrobot,
Tecnai Bio‐Twin Spirit TEM, FEI iCorr, high
pressure freezing/freeze‐substitution instruments, cryo‐microtome and
FEG‐SEMs. The facility offers excellent training opportunities for electron
microscopy related techniques. The University of Minnesota’s Supercomputing
Institute (http://www.msi.umn.edu) offers an excellent computation
environment.


 Contacts

Inquiries and applications should be sent to: zhang...@umn.edu or
man...@umn.edu

[ccp4bb] Phasing with Many Monomers/AU

[Chris Fage]

Hello Everyone,

I am currently trying to phase a structure with an asymmetric unit
predicted to contain 20-24 monomers (space group P1). The native crystals,
while beautiful in appearance (see attached), only diffract to ~3.4-3.0
angstroms at best, and SeMet-derived crystals grow with poor morphology
(small needles). Also, based a fluorescence scan, I know that mercury does
not bind appreciably. Other than screening for a new space group, what
options might I have for phasing this many monomers at lower resolution? Is
there any real chance of solving the structure in this space group?

Thank you in advance for any suggestions!

Regards,
Chris
<>

[ccp4bb] Prof Judith Howard to give a Friday Evening Discourse on crystallography at the Royal Institution

[Curry, Stephen]

Prof Judith Howard will be giving a traditional Friday Evening Discourse on

"A century of symmetry discovered: a crystallographer’s tale"

at 8 pm on Fri 31st January at the Royal Institution in London.

For those who might be interested, details of how to book can be found at: 
http://www.rigb.org/whats-on/events-2014/january/public-a-century-of-symmetry-discovered

Re: [ccp4bb] seeding, reproducibility

[Patrick Shaw Stewart]

One or more ingredients in the original hit (which you use to suspend the
seed crystals in) can indeed be essential, and may be all you need to get
nice crystals.  In D'Arcy's original paper you will see that he needed Ca2+
to get nice crystals of one of his proteins.  The same is true in Stoddards
paper, which inspired D'Arcy, ref below.  However in the majority of cases
it is just the seed that you need, i.e. there was a nucleation problem in
the new hits that appear with seed stock.  You can see this very clearly
because (in most cases) diluting the seed stock in the same solution gives
a corresponding reduction in the number of crystals.


Ireton, Gregory C., and Barry L. Stoddard. "Microseed matrix screening to
improve crystals of yeast cytosine deaminase." *Acta Crystallographica
Section D: Biological Crystallography* 60, no. 3 (2004): 601-605.



On 18 January 2014 00:18, Mahesh Lingaraju  wrote:

> Thanks you for the suggestions, Patrick and Matthias. I was actually
> wondering if any of the components from the seeding solution actually were
> important but your explanations sound more logical.
>
> I apologize for the large attachment. I did not realize that it was so
> big.
>
> Have a nice weekend !
>
> Mahesh
>
>
> On Fri, Jan 17, 2014 at 6:18 PM, Patrick Shaw Stewart <
> patr...@douglas.co.uk> wrote:
>
>>
>> Mahesh, this is a very interesting and slightly controversial question.
>>
>> One approach is to mix together all of the crystals that you have in the
>> initial screen.  The idea at the beginning of the project is to get as many
>> diverse hits as possible - you can worry about crystal size, space group
>> and quality later on.
>>
>> If you can collect data from crystals and determine the unit cell then
>> you can be more rational.  You can construct a "library" of polymorphs
>> having different unit cells.  This *may *allow you to push the space
>> group in a given condition to the unit cell or space group that you want -
>> maybe you find e.g. that your ligand can only diffuse into the active site
>> with a certain unit cell.
>>
>> There is a very interesting paper with several examples of how to use
>> polymorphs by the Stura group, see below.
>>
>> However, seeding can give rise to crystals with different but related
>> space groups.  A very nice example is shown in the wikipedia article about
>> epitaxy - titanium oxide growing on iron oxide.  Also Stura's paper on
>> epitaxial jumps is interesting and relevant.
>>
>> Make sure you dilute your seed stock in a systematic way as part of your
>> final optimization - one great advantage of microseeding is that oyu can
>> control the number of crystals per drop by diluting the seed stock.
>>
>> Also, make sure that you use fresh crystals to make the seed stock.  For
>> some strange reason old crystals sometimes fail to act as seeds, even
>> though they can be crushed and still diffract.  Maybe the unit cell
>> shrinks, or maybe the crystals become cross-linked.  Make the seed stock as
>> soon as your crystals stop growing, then freeze them.  Seed stocks can
>> almost always be frozen.
>>
>> Best wishes, Patrick
>>
>>
>>
>> *Library of polymorphs:*
>> Vera, Laura, Claudia Antoni, Laurent Devel, Bertrand Czarny, Evelyn
>> Cassar-Lajeunesse, Armando Rossello, Vincent Dive, and Enrico A. Stura.
>> "Screening Using Polymorphs for the Crystallization of Protein–Ligand
>> Complexes." Crystal Growth & Design 13, no. 5 (2013): 1878-1888.
>>
>> Stura, Enrico A., Jean-Baptiste Charbonnier, and Michael J. Taussig.
>> "Epitaxial jumps." Journal of crystal growth 196, no. 2 (1999): 250-260.
>>
>>
>> *Cross-seeding:*
>> Obmolova, Galina, Thomas J. Malia, Alexey Teplyakov, Raymond Sweet, and
>> Gary L. Gilliland. "Promoting crystallization of antibody-antigen complexes
>> via microseed matrix screening." Acta Crystallographica Section D:
>> Biological Crystallography 66, no. 8 (2010): 927-933.
>>
>> Abuhammad, Areej, Edward D. Lowe, Michael A. McDonough, P. D. Shaw
>> Stewart, Stefan A. Kolek, Edith Sim, and Elspeth F. Garman. "Structure of
>> arylamine N-acetyltransferase from Mycobacterium tuberculosis determined by
>> cross-seeding with the homologous protein from M. marinum: triumph over
>> adversity." Acta Crystallographica Section D: Biological Crystallography
>> 69, no. 8 (2013): 1433-1446.
>>
>>
>> *Seed stability etc*
>> Shaw Stewart, Patrick D., Stefan A. Kolek, Richard A. Briggs, Naomi E.
>> Chayen, and Peter FM Baldock. "Random microseeding: a theoretical and
>> practical exploration of seed stability and seeding techniques for
>> successful protein crystallization." Crystal Growth & Design 11, no. 8
>> (2011): 3432-3441.
>>
>>
>>
>> *Original description of the "random" microseeding method*D'Arcy, Allan,
>> Frederic Villard, and May Marsh. "An automated microseed matrix-screening
>> method for protein crystallization." Acta Crystallographica Section D:
>> Biological Crystallography 63, no. 4 (2007): 550-554.
>>
>>
>>
>>
>>
>>
>> On 17 January