X-ray Crystallography - Science method

Solving the structure of a protein that is mostly unstructured can be challenging, but it's not impossible. Here's a general approach you could consider:

Identify Structured Regions: Since you mentioned that only the part of the protein involved in binding to a specific component is structured, start by identifying and characterizing this structured region. This might involve experimental techniques such as X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy (cryo-EM).

Construct a Model: Using the known structure of the structured region, along with computational modeling techniques, try to construct a model of the entire protein. This could involve methods like comparative modeling (using known structures of homologous proteins), threading (comparing the protein sequence to known structures to predict its structure), or molecular dynamics simulations.

Experimental Validation: Once you have a model of the entire protein, validate it experimentally. This could involve techniques like small-angle X-ray scattering (SAXS) to characterize the overall shape and dimensions of the protein or hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe its dynamics and folding.

Complex Formation and Crystallization: If the protein forms a homodimer when binding to its component, you could try to form and crystallize this complex. This could potentially provide additional structural information about the unstructured regions of the protein, especially if they become ordered upon binding.

Yes, that’s correct! In strand displacement synthesis, the primer strand is extended using the template strand, and each nucleotide incorporated displaces the third strand that was annealed initially to the template.

The third strand is a short, single-stranded DNA annealed to the template strand and acts as a stabilizing agent for the displaced strand . The displaced strand is the second strand of the double-stranded DNA being synthesized.

More at:

- Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB | Nucleic Acids Research | Oxford Academic (oup.com)

-D-loop - Wikipedia

Jamal Nasir yes I would still confidently suggest there is excellent agreement. This is for several reasons (including more than 3 decades of experience), the main ones being that (1) we are comparing apples and oranges here - the TEM samples a relatively small number of grains, whereas XRD averages a huge number, and most important (2) the XRD-derived crystallite size is a simple calculation based on a simple model that rarely applies to real materials, and so I would suggest that if you think your XRD size error is "between 3 and 8%", then you need to think again - that would imply that the "error" for your examples, i.e., 6-8nm crystallite size, is +/- 0.2nm, which is a statistical error based on least-squares fitting parameters and is not actually the probable error in crystallite size, which is almost certainly an order of magnitude larger, i.e. +/- a few nm.

I think a lot of cell biologists could look at their cells under a microscope and immediately tell if their cell culture was contaminated, although they might not be able to identify which type of contamination. Trying to distinguish which type of contamination could be difficult. Personally, I don't think that microbiologists would be interested in the application. There might be more interest from biologists who study non bacterial eukaryotic cell types like HEK293, Hela, cancer cells, and other types of mammalian cells.

Size of prokaryotic cells is about 1 - 5 microns and size of eukaryotic cells is at least 10 microns and larger. On the basis of size at least by eye, it is easy to separate eukaryotic cells from prokaryotic bacterial cells.

Bacterial cells can be groups by size and morphology. They can be spheres, rods, curved rods, etc. and grow in isolate, chains, or clusters. That to some degree tells you if you are looking at Escherichia coli, vibrio cholera, staphylococcus aureus, etc, although false positives are possible because some bacterial cell types have the same morphology and growth pattern.

A lot of mammalian cell culture biologists complain about Mycoplasma contamination because the size of the mycoplasma bacteria is smaller than 1 micron so biologists might be interested in a application that could identify mycoplasma contamination. But cell biologists would probably look under the microscope immediately see the mycoplasma contamination because it is such a problem.

ImageJ is pretty standard for analyzing biological images. It is very powerful and you can write scripts for it, but it is kind of easy, but I don't think it is already automated for tasks like this.

When microbiologists complain about contamination, it is viral contamination of bacteriophages that they are complaining about. Bacteriophages are like nanometers in size so they can't be seen with a microscope but evidence of contamination is detected by lack of microbe cell growth due to lysis and the lysis of bacteria cells I believe you could detect from the images. Maybe microbiologists would be interested in an application that could detect lysed bacteria cells.

For this, please check the article, DOI: 10.13140/RG.2.2.27720.65287

These values are available in wwPDB validation reports (e.g., https://files.rcsb.org/pub/pdb/validation_reports/dt/1dtj/1dtj_validation.cif.gz), a snippet is given below.

Hi Ganesh, I´m afraid you can´t do that with PyMOL only, you need coordinate files for the open and the closed conformations, that should be obtained experimentally (eg. through X-ray crystallography, Cryo-EM, etc.). Alternatively, you could run simulations of the open-closed dynamics with some molecular mechanics software. But if all you want is to make a figure that *just shows* how the conformational change *would be* according to what you *think* it is, you can use the "drag" function of PyMOL, which allows you to manually move/rotate any group of selected atoms. Of course that would be a *fake* conformational change, only for illustrative purposes!!

CryoEM

You have two ionizable hydroxy groups in your enantiomers. Your compounds can also exist in a keto- and an enolic forms. Provided that you really have two pure enantiomers 1 and 2, the chemical shift of C7 and C8 must depend on the state of your compounds in solution and hence on the specific conditions used. Small variations in concentration of an acid or a base may change either the predominant form or the state of equilibrium in which your compounds exist. It should be very important to use the same purification procedure for each of the enantiomers and the same lot of solvent for NMR. One simple test you can do is to record an NMR from 1:1 mixture of 1 and 2. If your compounds are enantiomers, you should see only one peak for each C7 and C8. If the spectrum of the mixture shows two peaks for each C7 and C8, then your compounds are unlikely to be enantiomers.

As long as you have reliable CIF file - either software is fine. I personally used TOPAZ

Dear Jun Young Park ,

before trying to help you, there are some important questions that one needs from you.

Firstly, what's the resolution of te data, how did you solve the phase problem, and in what stage of the refinement are you? All of these are very important issues because an Rfree of 44% is way to high.

Secondly, regarding the screenshot itself, at what sigma level did you got it? The map seems very noisy, and any information at a very low sigma level, together with an Rfree of 44%, is not worth interpretation from the crystallographer.

Thirdly, I apologise but I cannot see any FoFc density in the screenshot. There is a very small green blob at the lower right corner but apart from that I cannot see anything.

I hope you can solve your problem and I apologise if I was not very helpful. Please contact me in private (or by e-mail: jbrito@itqb.unl.pt), if you need further assistance (or don't want to publicly divulge any details.

Best regards,

Jose

Dear Gabby,

if I recall correctly, SG 16 is P222 and SG 19 is P212121. So basically, what is happening is that the program is indexing and integrating in the Laue group primitive orthorhombic P222, but then, looking at systematic absences, it outputs space group P212121. Nothing wrong or cumbersome here.

If you want to process with XDS in lower symmetry, here's what you should change in your XDS.INP file:

SPACE_GROUP_NUMBER= 16 UNIT_CELL_CONSTANTS= a b c alpha delta gamma

Anyway, I take this opportunity to make a shameless advertisement (although I am not part of the team or have any interest in this advertisement whatsoever): try to process your data with autoPROC (https://www.globalphasing.com/autoproc/). autoPROC is a pipeline of programs that uses XDS, POINTLESS, AIMLESS and STARANISO for data reduction, space group assignment and iso/aniso scaling, respectively.

The program is quite visual and intuitive, and it will tell (you will see it!), what is happening with your data and what you can do to test different space groups or try different protocols to improve statistics.

Anyway, coming back to the beginning, if you are phasing by molecular replacement, you can use your P222-reduced dataset, and ask the MR program (usually PHASER), to try "all possible in same point group). This way you will know for sure if it's P222 or any of the subgroups of it.

Please contact me in private if you need further assistance.

Best regards,

Jose

Dear Cheng Yun Wu many thanks for sharing this very interesting technical question with the RG community. It might be worth a try using acetonitrile to recrystallize your ionic ruthenium complexes. Often such compounds show a good solubilty in hot acetonitrile and form nice crystals upon cooling of the solution to room temperature. Hot water alone might also be worth a try. If your compounds are very soluble in acetone, you could also try a vapor diffusion technique in which you let diethyl ether diffuse into the actone solution. For more potentially useful suggestions please also have a look at the attached guides to growing single crystals.

Good luck with your experiments and best wishes, Frank Edelmann

Crystalmaker X is compatible with mac OS and macbooks with the Apple chip.

X-ray-based structures are preferred because of their higher resolutions

Dear Jane, as a synthetic chemist I'm not an expert enough to give you a qualified answer to your interesting technical question. However, I just came across the following potentially useful article which might help you in your analysis:

Fortunately this article has been posted by the authors as public full text on RG. Thus you can freely download the pdf file.

Good luck with your research and best wishes!

Suggested Readings

Dear Shafikul, many thanks for asking this very interesting and important technical question. Growing single-crystals suitable for X-ray diffraction is often a more difficult task than preparing the respective compounds. I assume that you are equipped to handle air-sensitive compounds. The most common crystallization method is cooling of saturated solutions in organic solvents in the refrigerator or carefully layering such solutions with a non-polar solvent. Vapor diffusion techniques can also be tried within a glovebox. You can find a number of very useful crystallization guides in the internet. For example, please have a look at the attached articles.

Good luck with your work and best wishes!

Hi every one

I can do qualitative & quantitative analysis of XRD data for free.

Just send me your XRD data file and your material phases or elements.

Dear Ranjuna,

You may also find useful to read some example from Teaching and Education Section of JAC:

Indra Kumari

In this video tutorial, I have explained in detail the d-spacing (interplanar spacing) and how to calculate it from the XRD data using OriginLab software. In the case you want to further ask about it, please do comment on the specific video, I'll respond to it shortly. I have provided the practice as well as calculations files here. Thanks

Probably the latest version does not match with the hardware of your system.

The problem for all kinds of EXAFS / XANES simulation is a correct and physically valid structural model. This not only includes the positions of the atoms, but also disorder parameters, site occupancies, and potential models. Here, in particular for XANES calculations, small changes may have a big influence on the calculated spectra. For EXAFS, the situation is more safe, as those type of calculations are much more straightforward ... see e.g. the review by John Rehr (Rev. Mod. Phys. 72 (2000) 621, DOI 10.1103/RevModPhys.72.621). Hope this helps, Dirk

Dear Tarique Hasan Shushmoy ,

Should I retake XRD data?

No, it is not necessary. However, any mismatch in the experimental and theoretical calculated point in the fitting will generate a significant influence on chiˆ2.

What conditions should I follow if my data is not perfect for refinement?

It looks ok for refinement; as you can see, there is no peak between, let say, 5 and 22 or 23; that part could be eliminated in a new experiment or just put as a restriction to not be fitted during the fitting process. Only that procedure will decrease the Chiˆ2 that will not consider those background points 5 - 20 2theta.

I am not familiar with the incorporation of vacancies.

You may try it releasing the occupation of the metal sites. Firstly, it will give some insight into cation vacancies; you may check the anion vacancy in the same way. Be aware that doing the procedure you may allow distortion in the structure needs critical evaluation after the fitting.

How would I take oxidation state in fullprof?

Usually, when you define the cation, in the PCR, the Ca atom is in sequence written as Ca2+; this will tell the program how many electrons must be taken.

I have tried Pseudo-Vogit axial digvergent as well. It did not improve much.

In principle, it tells you there is no stacking fault problem or high distortion into your sample's peak profile. Use this information with care, probing other possibilities.

To adequately use FullProf, it is necessary to do it with a known structure first, feel how the fitting undergoes after that expend some time in your real sample.

Best regards

WNM

Anwar A. El-Hamaky

X-ray people might be impacted more than NMR and cryo-EM people by AF2. It may replace all biophysical techniques for structural determination someday, IMHO, they advertise much better than academic groups for the things they've achieved and still a long way to go (like when Rosetta first introduced). Not to mention the missing pieces like non-natural amino acids, binding partners, dynamics, different circumstances in the cell, more or less scientists would like to validate the prediction experimentally. However, it is hard to argue with its valuable inputs and insights before wet bench works that cost a lot.

For BCC matrix and interstitials mechanical loss spectra can be measured to quantify amount of interstices. More info you can find in the paper:

I Agree With Piotr Zabierowski

If your previous refinement strategy worked better then stick to it. Just rerun the original refinement strategy with the corrected rotamers as input and see in phenix.refine maintains them better without fitting the weight terms.

Agnishwar Girigoswami very interesting article, I will this into my research, Thanks for sharing.

Very simple one https://app.visiblegeology.com/stereonetApp.html

Yes, you can do any test related to the capsid's antibody, If the antibody is available; or If Capsid protein is immunogenic, which helps to produce antibodies in the mammalian system. You can get antibodies after expression, purification of this protein (immunogenic) through any compatible or recommendable system.

Williamson-Hall plot is a method for determination of "mean values" of crystallite size and strain. If a high quality program for Rietveld analysis or for whole powder pattern decomposition supporting microstruce analysis is available this might be better than evaluation by a Williamson-Hall plot.

To provide a Williamson-Hall plot you need a scan covering a large 2theta range and a method for determination of the full width at half maximum B (fwhm) of many peaks of the phase you are interested in. The fwhms must be corrected for instrumental broadening. The easiest would be to substract the line width b of a distinct peak of a well crystalline refernce sample yielding Bcor = B - b. More relialable methods for correcting exist.

The Bcor values (fwhm in degree) are transformed to arc values Bkor', Bcor'(arc) = Bcor(degree) / 180° * pi. Then a plot of Bcor'*cos(theta) versus sin(theta) will be produced. The value of the slope provides the "mean" strain epsilon. Ref. e.g. . http:/pd.chem.ucl.ac.uk/pdnn/peaks/sezedet.htm.

Anisotropic line broadening, correct values of instrumental broadening and shapes of the peaks of your sample may make things more difficult. Please also check

Regards

Robert

Unfortunately I have run out of time for further experimental work. But thanks a lot for the insight and suggestions, they have been very helpful !! :)

Hi Mehdi,

Why don't you look up the textbook "Rietveld Refinement: Practical Powder Diffraction Pattern Analysis using TOPAS" by R. Dinnebier, A. Leineweber, and J.S.O. Evans? There are also somee tutorials on the internet.

Good luck,

Andrzej

If you are recording XRD pattern of same materials using same diffractometer of same mounting, peak positions will not change with changing scan rate. Intensity of the XRD data shoes inverse behaviour to the scan rate.

"3) And the target should not have missing residues; [how can I know about this criteria?]"

Compare the sequence downloaded from the pdb (SEQRES records) to the sequence extracted from the coordinates.

Do a 3D alignment of the potential template structures and look at it carefully - it gives you an idea of which parts of the structure are flexible.

If there is a lot of flexibility in the vicinity of the putative ligand binding site, you might consider using several different structures representing different conformational states.

By comparing free to liganded structures, you can assess whether there are conformational changes associated with ligand binding. If so, a liganded structure (with the ligand removed) is better for docking.

EXPO2014 (http://www.ba.ic.cnr.it/softwareic/expo/) is good software for structural determination and rietveld refinement. It is easy to use and has a good manual.

There are other software like FOX (https://fox.vincefn.net/), DASH (https://www.ccdc.cam.ac.uk/solutions/csd-materials/components/dash/), etc.

To me, this type of crystals is caused from lithium. You can incubate your protein buffer without protein to the same reservoir and see what's going on.

Certainly you can use PEG powder to prepare crystallization solutions. I did that back in graduate school and could reproduce crystals initially grown from commercial PEG solutions. The thing is home-made PEG solutions are often not as reproducible as commercial ones due to their high viscosity, so it might be difficult to optimize/reproduce crystals that are picky.

Dear Gabriela,

Your question is quite interesting despite a very wide use of His-tags and common acceptance of multiple results obtained with tagged proteins. There are at least three considerations that you should take into account and follow the advice of George (doing experiments with and without).

1) Location of the His-tag (including the linker) can influence folding.

2) Mobility of the tag can influence the dynamics of the protein, the dynamics of the substrates and dynamics of the products, therefore overall activity.

3) His-tag can influence metal ion load by spurious chelation.

You may ask how I got this prompts?

As a careful crystallographer I refined many His-tags in my own as well as many deposited in PDB proteins. They are routinely unrefined by a common assumption that they are mobile and not important. But that is a usual error of a confirmation bias. We get what we expect, by not doing the job properly.

The experience shows that 30-50% of the His-tags, I refined, were clearly visible with comparable ED (electron density) level to the protein. In almost all the cases the His-tags influenced the conformation of the protein in some small but sometimes very substantial manner. In several cases they were responsible for aggregation and the form of a crystal. In two cases (because of the closeness to the active site) they clearly influenced the activity.

Good Luck.

Bog

Generally speaking; (I) the peak height or the maximum intensity in XRD spectra represents an approximation for the peak intensity, (II) the peak area a.k.a. the integral intensity can be considered as the real measure for the peak intensity that is related to the crystal structure that is commonly used to determine the amount of a particular phase, while (III) peak width or Full Width at Half Max (FWHM) aka half width represent crystallite size, and defects in form of strain and disorder (with line asymmetry associated with particle size). More detail description could be find at: http://www.fhi-berlin.mpg.de/acnew/department/pages/teaching/pages/teaching__wintersemester__2015_2016/frank_girgsdies__peak_profile_fitting_in_xrd__151106.pdf

Also, an example for the application of Rietveld method used in application of XRD in phase analysis can be seen at: http://www.icdd.com/resources/axa/vol47/v47_40.pdf

The two methods of structure determination are based on completely different properties of proteins. An NMR structure is calculated from magnetic properties of several nuclei while an X-ray structure is derived from electron density of non hydrogen atoms.

Structures of both methods have a high confidence, the determination of secondary structure elements, their relation and loops playing role in catalytic activity is reassuring. The possibility of some catalytic reactions in the circumstances of measurement reinforces the confidence of 3D structures. Regions, weakly defined by NMR or possibly affected by crystal packing, are determined less confidently. These are usually longer surface loops, chain terminals or domain interconnecting loops that have flexible conformation in natural circumstances as well. Pockets with catalytic activity and the framework of secondary structures that is responsible to fix the pocket are usually stabile enough not to be affected by the changes of circumstances.

The nature of the two methods results in the fact that NMR structures are never so concrete as X-ray ones, they allow larger freedom for motions of loops and terminals. It is probable that this freedom is related to dynamism of these regions in solution, however the modelling character of molecular dynamic calculations reminds to carefully handle such comparison.

Very useful, thanks!

With the availability of the purified proteins, you can consider various biophysical approaches, depending on what equipment is available, including surface plasmon resonance and isothermal titration calorimetry. Chemical crosslinking can also be a useful technique.

Dear Aga Shahee

I think the theory annunced buy Fewster’s is based on the misleading of the effect of solid surface structure which controls the basically the structure forms in the nanoscale sizes of crystals and may be appears clearly in the crystalline powder form. I had a quick looking to the article and he denoted to particles less than 10 microns for his hypothesis. In this size surface effetc will be significantly appears on the X-ray diffraction data for the powder form crystalline, for more details on nanosize crystaline state I suggest to see the following power point

"Solid Surface and Nanoscale materials Structure" M. S. Omar Presentation · July 2016 with 156 Reads DOI: 10.13140/RG.2.2.31947.59684 Charmo, University of Charmo, University of Salahaddin, DOI:10.13140/RG.2.2.31947.596

Hello Carisa... If u have the xpert high score plus software then first open the data file in the software...then go to treatment section and search peaks (u can do it by automatic mode or by selecting peaks manually) and click on accept. Then go to analysis section and go for search match....now before applying search go to restriction section...select the sub-file 'minerals'. if u know the probable mineralogy of the rock then in the restriction section select the possible elements in the periodic table. Now click on search tab. a set of possible minerals are appeared in the right tab of the screen. Now select the possible minerals and drop them in the upper pan and go for reitvield refinement for quantitative analysis.

Kindly note that this is the general process to get the quantitative values. For better results consult with some people who are frequently using this software (probably the chemistry or metallurgy student). they will guide u in a better way.

first of all i am extremenly sorry for late reply..

i was trying to ask that is strain depend on crystal structure ?if yes then if we have two structures eg barium titanate having tetragonal structure and barium zirconate having cubic structure .then in which compound we can expect more strain ....

i am expecting that now my question is not so confusing..

SNR to COD relationship for the above data set at location "800" on topograph.

Depends what you mean by "different"

Some XRD machines can be more sensitive than others, so if an older version of XRD machine shows mostly amorphous powder with no clear peaks, new machines can give you a good resolution of peaks, or show some peaks that older versions did not show. However, the strong peaks should be there with just different intensity. Ignore the intensity because it is arbitrary unit. The 2theta angle is more important and it should be the same.

It may also happen that the whole spectra is a little shifted to the left or right, this really happens because of the average height of the surface (for powder) is not at the recommended height, so you need to be careful to flatten the powder surface at exactly the height it must be. It is not easy to make flat surface at the right height if your powder is coarse. Grinding your powder, especially with a high energy ball milling technique can change the composition (adding impurities etc.), as was said above.

However I think that grinding in a mortar by hand should not generally change the peaks unless your powder can absorb humidity during grinding time, and change the powder composition.

thanks for your answer and for providing these references

I may add the following:  some proteins, especially membrane proteins, are exceedingly difficult to crystallize, but some researchers have solved this problem by genetically re-engineering the protein.  This would involve deleting regions that are known to be disordered, etc.  Thus when interpreting 3D crystal structures of re-engineered proteins, one should be very careful to consider the artificial changes that have been made to them.

open your  .lst file there you will find hkl values of the bad reflections, so copy these hkl values in .ins file and with omit command ...

This could be a concentration dependent artifact. Consider that a crystal of a protein is an ultra high concentration of protein - even though you set up the condition at say 10mg/ml the amount of protein molecules in a crystal (depending on size of the crystal and protein) could be >500mg/ml (or >50mM). If the crystallographic tetramer is symmetrical it is possible that the quaternary structure is real, but concentration or pH dependent. You can submit your monomer to the PISA server to calculate the probability of multimerization also. 

You can easily experimentally assess this phenomena with size-exclusion chromatography at different concentrations and pH as well as dynamic light scattering and/or Small Angle Xray Scattering. Regarding SEC, make sure you choose a column that is capable of resolving a tetramer and monomer for your studies. 

Also, you didn't mention the crystallographic condition (salt and concentration, etc), but salting out is something to consider when dealing with proteins in solution. If the condition includes 1M Ammonium Sulfate, then the nucleation could start with salting out the protein (for instance). 

Biomolecular Crystallography from Bernhard Rupp. Here is the link http://www.ruppweb.org/Garland/default.htm

Dear Kannan, 

I can definitely recommend a universal file conversion tool for the XRD files: PowDLL available at: (free)

it's free and offer conversion between many different file types used in XRD.

Best regards,

Sebastian

I'm not so sure about HSP V 2.0. But to do determine the amorphous phase if you know all the crystalline phases is by spiking method; add known wt% of crystalline material (not in the specimen candidates), do the quantitative analysis, change the refinement wt% of the crystalline material by its true wt%, in v3 u'll get the amorphous wt%. Or you can do POCNKS : "Quantification of phases with partial or no known crystal structure“ Nicola V.Y. Scarlett and Ian C. Madsen, Powder Diffraction (2006) Vol. 21(4), p. 278 – 284. doi.org/10.1154/1.2362855

Both are known as temperature factors or B-factors, may also be represented by U,  where B = 8π2 U.

Thermal motion and positional uncertainties may be isotropic or anisotropic. Anisotropic atomic displacement parameters are represented by 6 parameters; 3 variances of x,y and z and the 3 covariances of the possible pairs of coordinates.

This parameter must be done at the end or u'll get unreasonable results.

What is the resolution of you synchrotron data?

"2. Is it practically correct to refine a set of crystal structures from different research groups by TLS refinement to get a list of ADPs and compare them with the thermal or B-factors reported in PDB files?" the crystal structure is a model for you to fit your data.

You can find this information in manual, page 219

FOSQ observed F_o²

FOTSQ F_o² corrected for extinction and on scale of F_o²

FCSQ F_c² with scale and extinction applied

FCTSQ F_c² on absolute scale

Hi Mehri,

On youtube there are number of tutorial videos of Fullprof software, by which you can refine your xrd data. There is another software called Match, which will also help you to find the phase or structure that best matches with your xrd data. Can you share the compound name which you are trying to prepare or tell the starting materials and the expected final product name.

Thanks for the explanation of S. R. de Lazaro and Alexander B. Missyul’. Both of your answers are useful for me to further understand the growth mechanism of CdS particles  during preparation. Since we would want to know the preferentially grown planes and the atoms distributed on it, then we can judge the charges of each plane. Combining the pH condition of solution, we might able to deduce the effect of pH value on the preferentially grown planes, so as to the morphology of final CdS particles.

Thanks again for your explanation and concern. : )

Kun Li 

Also try GSAS-II, which is free.

Cool! I am happy to hear it!

Rizky! "the most popular equation model of preferred orientation is March-Dollase model", that is more than I know. I know a lot less than that I need to know. Thanks for the education :-)

I hope someone else in this erudite group can come up with the answer to your question so that I may learn as well. However, there is software similar to Bruker LEPTOS that allows folks to incorporate the latest theoretical models to simulate various linear diffractograms in order to compare with experimentally recorded diffractograms. It is critical to incorporate the "parameters" correctly to avoid errors and equivocation while using such simulation tools.

Hi Mohammed. It would be helpful if you include the question signs in your questions.

In order to extract the instrumental profile from your measurements you need to measure a free-of-strain sample first, with a crystalline size small enough to get a good statistic. There's a commercial sample, LaB6, you can get from NIST in the USA. It's very expensive but good to do the job. Another alternative would be preparing a powder from a single crystalline Si wafer, but it's not easy to do without applying some stress. If you can find a way to get rid of the strain in your sample, let's say, to a value below 10^-4, that could be useful as a reference for the instrumental profile.

Once you've measured your reference sample for the instrumental profile you fit the powder diffraction pattern in Xpowder and you store all the values of the reference pattern, which basically consist of the parameters of the Caglioti's formula. Then, the instrumental profile can be extracted from any other sample with a strain higher than the strain in the reference material.

If you change the set-up in the X-Ray diffractometer, the Caglioti's parameters have to be measured again.

You can't get the dislocation density from Xpowder directly. For that purpose I would suggest to take a look at an academic software developed by Mateo Leoni, in Italy. It's called PM2K. You can send an email to him to get a copy of the software.

If you want to develop your own code in Mathematica or Matlab to determine the dislocation density and crystallite size by Warren-Averbach method I can give some directions to get started, just send me an email to hf278@exeter.ac.uk

Best regards.

You should check the original source of those cards, a lot of conditions produce changes in apatite structures (pressure, °C, substitutions, doping, ...), but the database will not inform you about those conditions

You may simply want to attempt further (wide) screening to find additional crystal forms. I would also advise you to do additive screening (e.g. using the Silver Bullets additive screens). I have had a lot of success improving resolution via additives.

In situ proteolysis (addition of trace amounts of protease to your xtallisation experiment) has also been reported to be successful at finding new crystal forms with improved diffraction quality. You can read up on it here:

Another fast and efficient way of potentially improving your resolution is protein surface engineering in the form of lysine methylation (basically to reduce surface entropy). You can read up on this here:

The above methods won't require you to go back to cloning. However, stepping back to reflect on your construct is often best. You may want to introduce mutations that improve crystallisability through surface entropy reduction. Have a go at this server if you want:

Finally, use disorder prediction software to check if there are flexible regions on your protein which you can possibly remove.

Happy screening!

Thank you all for comments. I will get back to you when I will have more data on this complex.

First, are you sure that you don't have structure factors in your cif file?  That has been the common practice now for a couple of years.  However, even without the hkl values, you can use a GUI such as OLEX2 with SHELX to add H atoms at caculcated positions.  You can just open the .cif file in Olex2, select the atom and type HADD.

young people try to get quick answers for their problems with out putting much effort and they end up in social media with silly questions.

But the so called pseudo intellectual sales representative try to offer them a beautiful wife at the stake of ailing mother, rather than giving them what they require.

Dear RUMU try to spend some time with the software, you will figure out your self how to work with it or else there are plenty of other open source software's which are better than what you are using for the same analysis. The file format that the software required may not be the same as your data file, check for it.

Rietveld refinement has been introduced for structure refinement and only later it has been extended to quantitative phase analysis. If you enter Rietveld or better full-pattern refinement and XRD at google, bing, yahoo or whatever, you should get hundreds or even thousands of hits describing many different application examples. There are even several free software packages based on the least-square refinement (which is the basis of Rietvelds refinement and therefore no magic!) All mathematical assumptions like peak profiles, asymmetries, geometrical diffraction models etc. are limited since they reflect "never" real practical diffractograms. Therefore, the final decision whether a fit is reliable or not, you have to make. You can only read papers, judge, learn and decide what authors made wrong, how reliable their results are, or which idea is really suitable for your specific case. All software packages have some automatic mode which hopefully prevent to crash the refinement but the result is perhaps not very trustful and you have to play and learn and try to understand what is going on and which parameter affect what. Each software is a "stupid" least square refinement often optimized for a very specific problem. You have to find by yourself what do you need and what fits best. Only for this task you will need months or years. It is better, to ask more concrete questions. Commercial systems only want to give their customers a tool for a first analysis. And it mainly should impress how big is their competence. However, the better software is usually the free one since there are the guys who develop new approaches. XRD companies commonly "react" on the development and implement new and successful code later, when scientist checked their reliability. 

In order to learn how much a parameter will effect your pattern, the theoretical simulation will help more that starting with experimental patterns. You can simulate patterns and reload them as experimental signal and try to refine them. Then you learn more than speculating what some residual intensities may mean... You will perhaps find that you can describe the same patterns by different parameter settings which tells you that there is often not only one good solution which brings us back to my assumption that you as scientist need to decide how far you can go with your interpretation and when speculation begins... 

Dear Chethan,

giving a quick look at your input file and to your main problem ( the system doesn't go relaxing because of non-convergence of the energy), I have some suggestions that you can apply in order to fix (hopefully!) your problem:

1) Looking at the PBE Ultrasoft pseudopotential for the Molybdenum, the pseudopotential file suggest to use a "ecutwfc" not less than 48 Ry (where you use 50, which is potentially ok!), but also a "ecutrho" no less than 407. By default "ecutrho" is 4 times ecutwfc (200 Ry in your case, which probably affect negatively the convergence of the energy!): I suggest to increase both "ecutwfc" and "ecutrho" (not specified in your list) to 60 and 430 Ry. The carbon atoms should not be a problem.

2) try to reduce the value of the degauss from 0.001 to 0.01. It will make faster the running with a sufficient good precision.

3) the energy threshold for the convergence "conv_thr" I guess it's too high for having good realistic results. By default is set 1.0E-06. At least, follow this default setting for the convergence.

About the rest, for example the crystal structure, I don't know so much about this compound. I know that there are two phases, alpha and beta with respectively orthorhombic and hexagonal lattice. So, if you know the geometry of the system and the right atomic disposition, you can try to set an appropriate value for "ibrav" and insert the unit cell parameters.

I hope this can be helpful.

Best,

A.

The easiest way might be to use a polarizing optic, e.g. a petrographic microscope as these are usually polarizing microscopes. If your university has geologic faculty than they should own such a microscope. Assuming the material is translucent just prepare a thin section (~ 25 microns thick), place it on the sample stage and cross the polars. By rotating the stage the birefringence should sho up (4 times dakr, 4 times bright by 360° rotation) if there is any. From the interference colour you can at leat judge the amount of birfringence.

If you need the absolute value it turns out a bit more complicated, as you need a more special equipment. But any university with a petrologic (mineralogic) or crystallographic chair should own the equipment (more or less).

Hope it helps

Cryoprotectants, especially sugars like trehalose, are used to help stabilize proteins during lyophilization. Glycerol is also useful as a stabilizer, but it can't be used for lyophilization because it is a non-volatile liquid. It can be used as a stabilizer when freezing the protein for storage, but you will probably have to remove it before setting up your crystallization trials. The same goes for trehalose or other cryoprotectants.

If your protein is sticking to the concentrator, try using one with a different membrane chemistry. Another way to concentrate a protein sample is to put it in a dialysis bag and immerse the bag in a concentrated PEG solution, or bury it in a solid water-absorbent substance like carboxymethylcellulose. Still another method is to bind it to an ion-exchange resin, then elute it in a small volume of a high-salt buffer. Obviously, this leaves you with a salty sample. If the protein is His-tagged, you can do the same thing with nickel resin, eluting with a high concentration of imidazole.

Dissolve one crystal of each type in SDS-PAGE sample buffer, run them side-by-side on a gel, and see if one has a higher molecular weight. Or take diffraction data and compare the electron density maps.

Thank you very much everyone. We understood, it was not correct. Now we are getting the structure correctly. Many many thanks for your expert opinion.