ALMN

From: REX PALMER
Date: 9 September 2011 16:20

Dear CCP4'ers

In the input instructions for ALMN it says:

FIND peak maxpek [RMS] [OUTPUT filename]

Read peak threshold and maximum number of peaks. MAXPEK is the maximum number of peaks to find (default = 20). Up to MAXPEK peaks above PEAK will be found, and all symmetry related peaks generated.

If the keyword RMS is present, then the peak threshold is PEAK * RMS density, otherwise PEAK is the absolute threshold in the scaled map.

Question: RMS density of what and what is the formula used?

Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com

----------
From: Ian Tickle


Hi Rex

I would assume it's the RMS deviation of the rotation function value from the mean.  If so then it's defined in the same way as the "population standard deviation" as given here: http://en.wikipedia.org/wiki/Standard_deviation

Cheers

-- Ian

----------
From: Ian Tickle

Rex, sorry, addendum to my last post: the grid points in the Eulerian space of the cross-RF (which is what I assume you're talking about) do not occupy equal volumes in angular space as they would do in a Euclidean space (e.g. a normal electron density map).  For example the volume of any grid point on the beta=0 & 180 sections is zero (these sections actually each consist of a line of points, and a line of course occupies no volume).  Therefore it's necessary to weight the values in both the mean and the RMSD by the volume of each grid point (= sin(beta)).  Whether ALMN actually does this or not would require an examination of the source code, though it's probably quicker to ask Eleanor!

Cheers

-- Ian

----------
From: Eleanor Dodson
Yes - the RMS for the map is weighted by  sin beta

Here is the comment:

C Mean & RMS need to be weighted by sin beta, to allow for distortions
c     in Eulerian space.
c     On the last section, if beta = 90, the weight is 1/2 because of
c     symmetry. Here we only need to test for last section, because
c     (a) if last section = 180deg, the weight is zero anyway
c     (b) if the last section is not 90 or 180, the rms is not valid anyway

Eleanor

JRH input Re: [ccp4bb] Neutron data collection

From: Jrh <jrhelliwell@gmail.com>
Date: 23 September 2011 10:15

Dear Rex,

These issues of energy overlaps are addressed in theory, for either diffraction probe, in Cruickshank, Helliwell and Moffat 1987 Acta Cryst, and also by the same authors in 1991 Acta Cryst for spatial overlaps,  and in practice in eg Ren et al JSR 1999 and Nieh  et al JSR 1999. Basically the predominance of singlet reflection Laue spots is a consequence of the probability of prime numbers and, where you do have energy overlapped spots, the effectiveness of energy overlaps' deconvolution arises where you have symmetry equivalents and/or multiple occurrences of the same hkl, which usually one does.  The low resolution reflections in particular have a higher probability of occurring in multiples and thus are mainly the ones that require the deconvolution of intensities ie of the fundamental and its harmonic(s). The extracted intensities so obtained are actually of a very good precision. A high completeness through all the resolution range is overall readily achievable with Laue.

Re point 2. These issues, and advantages,  are explored in Blakeley et al 2004 PNAS which features freezing of such large crystals through to protein and ordered solvent, which there is more of, model refinement. Losses of diffraction quality for freezing attempts with bigger crystals in our experience is worse though ie more probable than with small crystals, and so if you don't have some sort of supply, would certainly be off putting, but obviously it's doable. Also it is my belief that as experience grows so will such procedures improve and the scope thereby widen, encompassing for example freeze trapping studies with neutrons as probe.

Greetings,

John

Prof John R Helliwell DSc 

On 21 Sep 2011, at 10:52, REX PALMER  wrote:

Re Neutron Data Collection:

1. What are the limits to data set completeness imposed by a Laue experiment versus those of monochromatic data collection?

2. What problems are caused by flash freezing the larger protein crystals used for neutron data collection which do not occur for X-ray data collection ie because smaller crystals can be used.

Any help will be greatly appreciated.  

Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com

Vacancy at PDBe for an enthusiastic junior annotator

Hi all,

The Protein Data Bank in Europe (PDBe; pdbe.org) is looking to recruit an expert structural biologist to join the PDBe curation team at the EBI near Cambridge, UK. Applicants should be computer-literate and possess a recent PhD in some area of structural biology or structural chemistry as well as a broad knowledge in molecular biology or biochemistry. An in-depth knowledge of protein structure (including structure determination, analysis and validation) is essential. A few years of post-doctoral research experience in structural biology, as well as hands-on experience in the determination of protein structure is highly desirable.

The ideal candidate will be familiar with Linux/Unix operating systems and molecular graphics software. Basic programming skills, e.g. with Perl, Python or Java, would be an advantage. Given the extensive interactions with colleagues in the PDBe team as well as with international collaborators, depositors and users, excellent written and oral communication skills, fluency in English, ability to work in a team and attention to detail are required.

More details and a link to the electronic application procedure can be found here:

http://ig14.i-grasp.com/fe/tpl_embl01.asp?s=MbkMjPUrEcTFkHhTcz&jobid=46332,8998483234

Feel free to pass this on to suitable candidates!

--Gerard

PS: Some hints for prospective applicants: it doesn't hurt to add a nice cover letter in which you explain why you would love to be a PDB annotator and why PDBe strikes you as a brilliant place to work. Also, if you should get invited for an interview and we ask you what the main colour of our website is, please don't answer "blue"...

---
Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK
Secretary: Pauline Haslam  pdbe_admin@ebi.ac.uk

Beamline Scientist position available for small- and wide- angle x-ray scattering beamline at NSLS-II

The Photon Sciences Directorate at Brookhaven National Laboratory is seeking an experienced scientist to lead the effort in the development of a small- and wide-angle x-ray scattering (SAXS/WAXS) beamline at NSLS-II, which is a new third-generation synchrotron facility being constructed on Long Island, New York, with extremely high brightness and exceptional beam stability over a wide spectral range.  As the Group Leader, the selected candidate will lead a group of scientific and engineering staff to design, build, and commission a state of the art SAXS/WAXS beam line, known as High Brilliance X-ray Scattering for Life Sciences (LIX), and it's associated scientific programs at NSLS-II; please see http://www.bnl.gov/nsls2/beamlines/2010BeamlineProposal-Approved.asp for more information.  Responsibilities include interacting with the scientific user community to define the mission and technical scope of the beamline and managing all aspects of the beamline design, construction, and commissioning, including aspects associated with work planning and execution and cost and schedule performance reporting.

For more information and to apply, go to: http://www.bnl.gov/HR/careers/

Beamline Scientist position available for macromolecular crystallography beamlines at NSLS-II

The Photon Sciences Directorate at Brookhaven National Laboratory is seeking an experienced scientist to lead the effort in the development of a pair of canted macromolecular crystallography beamlines at NSLS-II, which is a new third-generation synchrotron facility being constructed on Long Island, New York, with extremely high brightness and exceptional beam stability over a wide spectral range.  As the Group Leader, the selected candidate will lead a group of scientific and engineering staff to design, build, and commission a pair of state-of-the-art macromolecular crystallography beamlines, known as (1) the Frontier Macromolecular Crystallography (FMX) beamline and (2) the Highly Automated Beamline for Macromolecular Crystallography (AMX) beamline.  Please see http://www.bnl.gov/nsls2/beamlines/2010BeamlineProposal-Approved.asp for more information.  Responsibilities include interacting with the scientific user community to define the mission and technical scope of the beamline and managing all aspects of the beamline design, construction, and commissioning, including aspects associated with work planning and execution and cost and schedule performance reporting.

For more information and to apply, go to: http://www.bnl.gov/HR/careers/

Postdoctoral Position High-throughput X-ray Crystallography/Structure-Based Drug Discovery at The Institute of Cancer Research

(B494) Postdoctoral Training fellow - High Throughput X-ray Crystallography/Structure-Based Drug Design
Cancer Research UK Cancer Therapeutics Unit and Division of Structural Biology, The Institute of Cancer Research  (Sutton, Surrey & Chelsea, London) 

The Institute of Cancer Research (a college of the University of London) is a world-class cancer research organisation with HEFCE RAE ratings of international excellence across all of its research programmes, including cancer drug discovery and development. In partnership with The Royal Marsden NHS Foundation Trust, we form the largest comprehensive cancer centre in Europe, dedicated to research that extends from epidemiology, genetics and molecular biology, through drug discovery and development, to cancer diagnosis and patient treatment.

The Cancer Research UK Cancer Therapeutics Unit (CTU), embedded within the Division of Cancer Therapeutics, is a multidisciplinary 'bench to bedside' centre, comprising around 160 staff dedicated to the discovery and development of novel therapeutics for the treatment of cancer. The CTU's exciting goal is to discover high quality drug candidates for validated biological targets and to progress these candidates to clinical trial. All the scientific disciplines are in place to make this possible, including medicinal chemistry, biology, drug metabolism and clinical specialists.

A postdoctoral position is available immediately in Dr Rob van Montfort's Hit Discovery and Structural Design Team within the CTU. The Post-doc will be involved in high-throughput X-ray crystallography and fragment-based screening and will be responsible for crystallisation and structural analysis of protein-ligand complexes from the CTU's drug discovery programmes. The successful candidate will interact closely with the biology, computational chemistry and medicinal chemistry teams at the CTU as well as with the protein crystallographers in the Division of Structural Biology in which the crystallography efforts of Dr van Montfort's team are embedded. Therefore the successful candidate will be expected to work across the two sites in Chelsea, London and Sutton, Surrey.

Applicants must have a PhD in a biological or physical science, and experience in macromolecular crystallography / crystallisation (to include protein biochemistry, protein crystallisation, & protein crystallography). Experience in molecular biology, protein purification, and/or structure-based drug design will be an advantage.

The starting salary for the position will be in the range £30,289 to £35,040 p.a. inclusive (based on previous post-doctoral experience) and the post is offered initially on a fixed term contract of up to 3 years. Informal enquiries to rob.vanmontfort@icr.ac.uk or isaac.westwood@icr.ac.uk.

Please DO NOT send your application to Dr van Montfort or Dr Westwood; CVs must be submitted in line with the instructions below.

To apply please email your CV and covering letter (addressing where you meet the person specification and including the names and addresses of three referees), to: recruitment@icr.ac.uk quoting reference number B494. For a job description and person specification visit www.icr.ac.uk or call our 24 hour recruitment line on 020 7153 5475. In a separate email, please send your completed equal opportunities monitoring form http://www.icr.ac.uk/jobs/current_vacancies/4959.doc  to the same email address quoting the job reference number above.

Closing date:  12^th October 2011

Dr. Rob van Montfort

Team Leader Hit Discovery and Structural Design

Sections of Cancer Therapeutics and Structural Biology

The Institute of Cancer Research

15 Cotswold Road

Sutton SM2 5NG

UK

Post-doctoral position for structural studies on multifunctional intrinsically disordered actin-binding domains, CNRS, in Paris suburbs, France

A 2-year post-doctoral position for structural studies on modular proteins containing multifunctional intrinsically disordered actin-binding domains is available at the Laboratory of Structural Enzymology and Biochemistry (LEBS), CNRS, in Paris suburbs, France. Starting date: October/november 2011. European funding - monthly gross salary: ~ 2 500 euros.

Project:

Structural basis governing the functional versatility of intrinsically disordered WH2 domains in actin assembly dynamics

Job description:

            The actin cytoskeleton relies on the ability of actin ATPase to self-assemble into polarized filaments which provide in all cells mechanical force for cell shape maintenance, traffic or membrane deformation. It forms a dynamic structural network constantly remodeled in non-muscle cells by many actin-binding proteins.

The postdoctoral research project study the structural basis governing the functional versatility of β-thymosin (βT) and WH2 domains in actin self-assembly dynamics. These small ubiquitous actin-binding domains are intrinsically disordered but functional with highly variable sequences. Found as single or repeated modules in numerous signalling modular proteins they appear to regulate very versatile functions in actin assembly dynamics [recent reviews: Husson et al. (2010) Annals of the New York Academy of Sciences 1194, 44-52; Carlier et al., (2011) Int Rev Cell Mol Biol. 2011;290:55-85].

We try to establish the structure-function relationship of βT/WH2 domains and the structural basis of their multifunctionality through a multidisciplinary approach integrating biochemical studies (rapid kinetics mechanisms of protein-protein interaction and self-assembly of actin), biophysical (hydrodynamic characterizations, epifluorescence microscopy in reconstituted motility medium) and structural (small-angle X-ray scattering in solution and X-ray crystallography). These techniques are used (1) to identify in vitro all existing activities in actin assembly found in different modular model WH2-containing proteins regulating massive actin assembly in cell migration, developmental processes or bacterial pathogen invasion, and (2) to understand how these functions are regulated by small sequence variations, their intrinsic flexibility, some arrangements in repeats or associations with other regulatory adjacent domains [Bosch et al. (2007) Molecular Cell 28, 555-68; Husson et al. (2011) Molecular Cell 43, 464-77].

            The laboratory LEBS at CNRS provides state-of-the-art equipment for protein expression, purification and characterisations by biochemical and biophysical methods. Crystallization robotics are available in-house and we have regular access to synchrotron sources.

Skills required:

 The successful candidate should have a PhD in biochemistry/structural biology/biophysics/biology with less than 3-4 years postdoctoral experience and strong practical skills in molecular biology and protein crystallography, with strong interests for multidisciplinary approaches on protein-protein interactions (www.lebs.cnrs-gif.fr/carlier/carliereng.html).

Location:
            The laboratory is located in a very pleasant green setting within the large CNRS campus of Gif-sur-Yvette which is 45 mn away from Paris via local railway (RER B). We benefit from a dynamic scientific surrounding, in close vicinity to Paris labs, Orsay University, the Atomic Energy Commissariat (CEA) center of Saclay and the SOLEIL synchrotron (5 min away by car).

Contact:

            Please send a CV, a short summary of your research experience and letters of recommendation of two referees to Louis Renault (renault_at_lebs.cnrs-gif.fr). I will be happy to provide further information to interested candidates.

Post-doctoral position in Biochemistry and Structural Biology with emphasis on Antioxidant Proteins

From: Karen Fulan Discola
Date: 20 September 2011 16:40

Post-doctoral position in Biochemistry and Structural Biology with emphasis on Antioxidant Proteins. University of Sao Paulo Sao Paulo, BRAZIL A postdoctoral position is available for research in structural biology of antioxidant proteins. The research also focuses on the mechanism of action of antioxidant proteins and on oxidative stress response. Our program encompasses a broad range of methodologies including: biochemistry, genetics, chemistry and pharmacy. Studies will be directed on different aspects of Cys-based proteins such as thioredoxin, glutaredoxin, peroxiredoxin and a new class of proteins named Ohr (Organic Hydroperoxide Resistance Proteins). More details and publications are available on 

www.ib.usp.br/antioxidantes. Interested candidates should submit a CV, two letters of recommendation and a statement of interest to:

Luis Netto (nettoluis@yahoo.com.br)

Department of Genetics and Evolutionary Biology Institute of Biosciences University of Sao Paulo

refmac and DNA

From: Ed Pozharski
Date: 8 September 2011 14:35
After switching (finally) to 6.2.0 and therefore to Refmac 5.6.0117 I
have found a problem working with DNA that I have not seen with
6.1.13/5.5.0109.  Namely,

- if I use the pdb file produced by Coot (0.7.pre-1.3470) that seems to
output DNA as Ad/Td/Gd/Cd no matter what the input names were, refmac
fails with the warning that it found a new monomer.  It appears that it
stumbles upon the very first thymidine, but in a strange twist it
reports the problematic residue having the name "DY"!

- if I use the pdb file previously produced by refmac, which has the
A/T/G/C as residue names, it fails too but now complains about the "new"
monomer named "T".

- the workaround I found is to rename all the thymidines to "DT".  It is
a bit annoying since coot keeps renaming them (well, not refmac/ccp4
problem per se) and I have to rename back (easily scripted task, of
course).  What is peculiar is that Ad/Gd/Cd don't need to be renamed
(does this have anything to do with thymidine being the only one that
changes residue name in RNA?).

Has anyone else seen this or it's something specific to my setup?

Cheers,

Ed.

--
After much deep and profound brain things inside my head,
I have decided to thank you for bringing peace to our home.
                                   Julian, King of Lemurs

----------
From: Miguel Ortiz Lombardía
Weird. I'm using Coot 0.7-pre1.3628 ( a bit ahead of yours but not so
much? or maybe yes? ) and DNA residues are saved in what I think it is
PDB v3 format (  DA, DC, DG, DT ) These files are properly understood by
refmac 5.6.0119, which at the end produces coordinate files in the same
format ( so DNA residues are:  DA, DC, DG, DT )

The only heck with coot/thymidines is that if you "add a terminal" T or
mutate another DNA residue to T, it lacks its C7 ( formerly C5M ) atom.
However, if you build an ideal T, it has C7... My workaround is to add
those pesky C7 atoms in dummy positions and refine.
Not what I find (see above)
Perhaps the Nd/DN issue was solved between revisions 3470 and 3628 ?

Best,


--
Miguel


----------
From: Ed Pozharski 

Indeed.  I just built the rev 3633 and it works fine.  Autobuild scripts
used to have some problems on Lucid, but this time it worked perfectly.

This evolved into a coot question, but I am still perplexed as to why
refmac would complain only about T and not A/G/C.

----------
From: Miguel Ortiz Lombardía
A, C and G are RNA nucleotides. T is (mostly) not, its RNA-equivalent is
uridine phosphate, U.

Cheers,

----------
From: Ed Pozharski
Right, that was my suspicion.  But I thought that RNA bases would be Xr,
not Xd.  Plus, refmac does not complain about missing oxygens.  Anyway,
the latest coot rocks, as usual, now if I only can figure out who is
Linda...

Cheers,

Ed.



--
Coot verendus est

----------
From: Miguel Ortiz Lombardia
Le 11/09/2011 00:23, Ed Pozharski a écrit :
My understanding is that the Xr/Xd scheme has been dropped from the
latest refmac5/coot to comply with the X/DX (and OP1, OP2, prime signs
instead of asterisks...) standard from the PDB/NDB.

Crystypos [WAS: [ccp4bb] Mac OSX 10.7 Lion]

From: Ed Pozharski

The best X-ray related typo I ever seen was the "Small angel scattering"
- poor little things!

On Fri, 2011-09-09 at 18:23 -0400, Patrick Loll wrote:
> Still doesn't beat my all-time favorite, an early Microsoft spell-checker that changed "diffract" to "defrocked."
>
> >
> > I forgot to mention how delightful the spelling auto-"correction"  feature can be.  (It should have read "nothing unusual in and of itself").
> >
> > That, at least, can be turned off.

----------
From: Vellieux Frederic

Well in fact, it all depends on the type of detector these small angels end up on and on the speed of this "godly" radiation. Only once you have considered both these elements can you say "poor little things".

My 2p worth.

Fred.

drops swelling

From: anita p
Date: 9 September 2011 12:22

Dear Crystallographers,
I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it.
This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition.
Could any one suggest a remedy for this.

with regards
Anita

----------
From: Poul Nissen
Yes - add glycerol to the reservoir -
15% glycerol has a huge influence on the vapour diffusion gradients
Poul

----------
From: David Roberts
The following is simply a quote from a recent post by Enrico Stura. Probably has a lot to do with what you are finding:


"Glycerol is also great to reduce nucleation. If you decide to add glycerol to the protein solution (for solubility, but in your case it might be for stability
reasons), you also need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and
not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context:
Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762.
http://pubs.acs.org/doi/abs/10.1021/cg101364m "

Good luck

Dave (but really, this is all Enrico)

----------
From: George T. DeTitta
Dear Anita

You are seeing the effects of a very modest reduction of vapor pressure of water with polymers. You could add salt to your reservoir and you could insure your drops are at the same temperature as your reservoir - not easy in conventional "constant temp" incubators.

George
Sent via BlackBerry by AT&T

---

----------
From: Enrico Stura
Anita,

see the message I posted yesterday on the CCP4bb:
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg22638.html

or google "stura glycerol"
Re: [ccp4bb] How to help crystal grow bigger

In your case the sentence you need is:
"You need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context: Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762. " Enrico.

--


----------
From: anita p
Thanks a lot for your suggestions.
Would it be worth while to try and remove glycerol at the last step which is gel filtration and then set trays in my case?
I have never tried without glycerol.
reg.
Anita

----------
From: Ed Pozharski

Sure, assuming that your protein does not become unstable without
glycerol (protein solubility is likely to decrease too).  On a general
note, protein crystallization is the (dark) art of trying as many things
as you possibly can (including the crazy stuff).  Quoting the great
Bruce Foxman, you need to use the crystallization method number eight,
which is the one that works :)

--
Hurry up, before we all come back to our senses!
                         Julian, King of Lemurs

How to help crystal grow bigger

From: SnowDeer
Date: 5 September 2011 09:06
Dear All:

Recently I am working on a protein which can already grow nice pyramid-like crystals after the condition was optimized, while the crystals are too small to be picked up. The crystals grew quite fast and densely, so I tried to put 100ul paraffin oil inside the 600ul reservoir solution or put the plate under 16 degree to slow down the evaporation, while the crystals were still the same. I also tried macro or micro seeding with or without the paraffin oil. Macroseeding would give a larger crystal (not very nice) with many small crystals in the drop even I washed the seeds carefully. For microseeding, the same small crystals grew.

I don't have many experiences in crystallography, so I have no idea how to make it grow bigger...

Any suggestion is most welcome.

Thanks.
SnowDeer

----------
From: Enrico Stura
dear SnowDeer,

The first step to see if bigger crystals can be grown is to use bigger protein drops and vary
the precipitant/protein ratio at the beginning of the vapour diffusion experiment.
You can work out for yourself why this should give you all the informations that you need in subsequent experiments.

Enrico.

--
Enrico A. Stura D.Phil. (Oxon) ,  
Room 19, Bat.152,                
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html

----------
From: ChenTiantian
Agree with Enrico.
Reducing the concentration of your protein sample to make it form less nuclei, then it might grow bigger.

Best R,

tiantian

--
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park,
Shanghai, 201203

----------
From: David Waterman
Dear SnowDeer,

Just how small is too small? If you have access to a microfocus beamline you might find you can collect decent diffraction data from crystals with dimensions in the single digit microns. Fishing tiny crystals is difficult, but something like the MicroMesh "tennis racquet" mounts can help. Having multiple crystals on the same pin is in fact a rather helpful way of screening lots of samples. Please don't discount your crystals _only_ because they are small!

Shameless plug: there is a review on microcrystallography here that you might find interesting.

http://www.tandfonline.com/doi/abs/10.1080/0889311X.2010.527964

Best wishes

-- David

----------
From: SnowDeer
To Boaz: I seperated the large crystals and checked its diffraction already. While the diffraction is quite poor, only several dots could be seen. T_T
I washed the seeds twice with my buffer and seed the drop immediately after setting it. Thanks for your advices and I will try the additives.

To Charles, Enrico, Bernie & Tiantian: Thanks for your kindly advices, I set different conditions for the buffers with glycerol and different protein/reservoir volume ratios already following your instructions. :)

To David: Hmm...my crystals are smaller than the smallest loop T_T. It's quite hard for me to pick them up (due to my clumsy fingers...lol). Thanks for your advices and the review.

I have another question: I usually stored my protein samples aliquots at -80 degree and thaw the small aliquots when I need to use. While my senior said it will harm the protein so she suggested to keep them at 4 degree. So it is possible that I got the small crystals coz the freezing and thawing alter the proteins?

Thanks very much.
SnowDeer 

----------
From: Enrico Stura
SnowDeer,

Additives are indeed a good idea, make sure you do it in an informed manner, each additive is different and you will
get a benefit only if you know how to do it right. I do not recommend a random approach.

For example, you mention glycerol. There is a lot to know on the subject and probably more to discover.
Glycerol will help freezing/thawing cycles and improve protein stability if you want to work at higher temperatures than
4°C. You should check increased stability effect out since it is easier to work outside a cold room.
I assume you are trying to set up your drops at the same temperature at which they will equilibrate. Which is a good thing to do.

Yet, plan your experiments carefully. Also look at previous messages on ccp4bb on the subject of glycerol
in particular regarding propanediol. Annie Hassell among others has posted some very good comments. This bullettin board
has been very keen on the subject in the past, so you can learn a lot from the archives.

Glycerol is also great to reduce nucleation. If you decide to add glycerol to the protein solution (for solubility, but in your case it might be for stability
reasons), you also need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and
not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context:
Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762.
http://pubs.acs.org/doi/abs/10.1021/cg101364m

Good luck, but most important work with precision. To go from small to big crystals you need to be very
precise on how you set up your experiments and understand the rate at which you equilibrate your drops.


Enrico.

----------
From: Brad Bennett
Hi SnowDeer-

What's your precipitant? Have you tried lowering that as much as possible? This will likely delay nucleation and crystal growth but the crystals will also likely be larger (and perhaps more well ordered). Of course, you may reach a lower limit where you prohibit nucleation. You'll have to determine this empirically.

How about smaller reservoir volumes while keeping the drop volume the same?

Have you tried setting up and storing trays at lower temperatures, 10 C or less? Sitting drop vs. hanging drop? Different oils in your vapor diffusion experiments, like Al's oil? 

Also, I had success with growing large crystals by setting up microbatch drops under oil. If you'd like details, I can provide them off-board.

Cheers-

Brad