Sumber: Wikipedia

Dalam genetika, microRNAs (miRNA) merupakan molekul RNA untai tunggal yang terdiri dari 21-23 nukleotida dan berfungsi dalam regulasi ekspresi gen. miRNA dikode oleh gen-gen yang ditranskripsi dari DNA tetapi tidak ditranslasi menjadi protein (non-coding DNA); akan tetapi diproses lebih lanjut dari transkrip primer yang dikenal sebagai pri-miRNA menjadi struktur stem-loop yang disebut pre-miRNA dan akhirnya menjadi miRNA fungsional. miRNA mature memiliki komplementasi parsial terhadap satu atau beberapa RNA messenger (mRNA), dan fungsi utamanya adalah sebagai downregulasi ekspresi gen.

Sumber: Wikipedia

Archaeologists and anthropologists worldwide have dug up plenty of skeletons over the years, but the bones seldom say much about where ancient peoples originally came from. Thus researchers have tried using variations in the genes of living individuals to trace their ancestries back to prehistoric times. In general, the closer two modern populations are genetically, the more likely that they share a common ancestry; yet this ancestral heritage is sometimes obscured by genetic changes that have taken place over thousands of years, as well as by interbreeding between populations. Happily, efforts to get around these complications have been boosted by an ever-growing mound of data about genetic differences between human populations.

A team led by geneticist Daniel Falush of University College Cork in Ireland developed a new mathematical model to compare not just individual genes or short DNA segments, as previous studies have done, but also very long stretches of DNA. Falush and his colleagues analyzed 32 DNA segments, each consisting of more than 300,000 base pairs, from 927 people representing 53 different populations from around the globe. Plugging this huge amount of data into computer simulations, the team worked out which migration scenarios were most likely to have created the genetic variation we see today. The results, reported today in PloS Genetics, suggest that modern humans peopled the world in nine phases, beginning in Africa, moving on to Europe and Asia, and finally colonizing the Americas and the Pacific islands. (The team illustrates humanity’s journey in two movies accompanying the paper; see below.) The team did not try to date the migrations.

The study came up with two unexpected findings. One is that the people of the Orkney Islands, to the north of Scotland, share some ancestry with Siberians, possibly because some ancestors of modern Orcadians ventured to Asia via the Arctic Circle. The team also found that North and South America were colonized independently by at least two different waves of migration from different parts of Asia, although both waves appear to have arrived via the Bering Strait. This conclusion contradicts the conventional view, which postulates just one migratory wave out of Asia.

“I like the paper very much,” says Jonathan Pritchard, a human geneticist at the University of Chicago in Illinois. “It’s a very novel and creative way of thinking about the data” that “may provide a better representation of human history.” Ripan Malhi, a molecular anthropologist at the University of Illinois, Urbana-Champaign, says that the team’s approach “holds great potential to give us important and novel insights into the peopling of the Americas.” Nevertheless, Malhi cautions that the multiple migrations Falush and his colleagues detect in the Americas might be an artifact of ancient population movements “more complex than the simple models created in this study can accommodate.”

Related sites

Watch human populations march across the globe in two movies created by Falush and his colleagues:

http://www.plosgenetics.org/article/fetchFirstRepresentation.action?uri=info:doi/10.1371/journal.pgen.1000078.s008

http://www.plosgenetics.org/article/fetchFirstRepresentation.action?uri=info:doi/10.1371/journal.pgen.1000078.s009

Oksitosin (berasal dari bahasa yunani yang berarti “kelahiran cepat”) merupakan hormon mamalia yang juga berperan sebagai neurotransmiter di otak. Oksitosin dibuat oleh sel-sel magnocellular neurosecretory dalam otak. Pada manusia, oksitosin dilepaskan oleh pria maupun wanita saat berpelukan, bersentuhan, dan orgasme. Pada otak, oksitosin berperan dalam interaksi dan hubungan sosial, dan kemungkinan juga terlibat dalam pembentukkan rasa saling percaya diantara manusia. Pada wanita, oksitosin dilepaskan dalam jumlah besar setelah pembesaran cervix dan vagina selama melahirkan, setelah menstimulasi puting susu, dan menyusui. Oksitosin sintetis dijual untuk pengobatan dengan nama pasar Pitocin dan Syntocinon.

Tikus merupakan hewan pengerat yang banyak hidup di rumah-rumah. Mereka biasanya memakan makanan yang di simpan di dapur dan atau merusaknya. Tapi, tau gak, ternyata air kencing hewan pengerat ini bisa sangat bermanfaat, disamping bahayanya terhadap kesehatan.

Krishna Persaud dari Universitas Manchester, UK, dapat memanfaatkan urine tikus sebagai pendeteksi  bau yang sensitif dengan memanfaat sifat-sifat khusus dari urin tersebut. Alat ini bahkan dapat mendeteksi polutant dalam jumlah sangat kecil sehingga dapat digunakan sebagai alat pengontrol kualitas makanan.

Kenapa air kencing tikus?

Ternyata, urine tikus mengandung protein urine utama dalam jumlah besar (MUP). Protein ini dapat menahan molekul bau dengan kuat dan melepaskannya perlahan-lahan. Tikus menggunakan trik ini intuk menandai wilayahnya.

Dengan menempelkan MUP pada microbalance kristal kuarsa, maka kita membuat alat sensor bau yang sensitif. Kristal yang digunakan pada alat ini dapat bervibrasi pada frekuensi tertentu ketika ada arus yang melaluinya. Jika massa kristal berubah, karena menempelnya molekul kecil pada protein di kristal, maka akan terjadi perubahan frekuensi yang dapat diukur. Alat ini memiliki sensitivitas sampi beberapa nanogram dan dapat mendeteksi molekul pada konsentrasi ppm.

Keni V.

Sumber: nature news, 9 May 2008

Tau gak, ternyata mendengarkan musik dapat mempercepat penyembuhan pasien yang terkena stroke. Berdasarkan hasil penelitian terhadap hewan, disimpulkan bahwa lingkungan auditori dapat mempercepat proses penyembuhan kerusakan syaraf. Diketahui bahwa mendengarkan musik dalam waktu lama memiliki dampak positif terhadap fungsi kognitif dan emosional para pasien yang menderita stroke arteri permukaan otak besar bagian kanan atau kiri.

Kesimpulan ini diambil berdasarkan pada hasil penelitian yang dilakukan oleh para peneliti dari Finlandia. Mereka melakukan studi terhadap 60 pasien dari berbagai rumah sakit di Helsinki dari bulan maret 2004 sampai mei 2006. Para pasien tersebut di bagi secara acak ke dalam tiga kelompok, yaitu kelompok musik, kelompok bahasa, dan kelompok kontrol. Tiap kelompok berjumlah 20 orang. Kelompok musik diminta untuk mendengarkan rekaman musik selama ≥ 1 jam per hari selama dua bulan. Kelompok pasien bahasa diminta untuk mendengarkan rekaman buku audio selama ≥ 1 jam per hari, juga selama dua bulan. Sedangkan kelompok kontrol tidak menerima materi rekaman apa pun selama masa penyembuhan dari penyakitnya. Semua pasien menerima perawatan rehabilitasi standar stroke yang sama. Berdasarkan hasil monitoring selama enam bulan, kelompok musik mengalami peningkatan yang pesat dalam kemampuan memori verbal dan konsentrasi dibandingkan kelompok bahasa maupun kontrol. Kelompok musik juga memiliki tingkat depresi yang paling rendah dibandingkan kelompok kontrol.

Jadi, mendengarkan musik setiap hari merupakan sarana rehabilitasi yang sangat murah tapi sangat berharga untuk penderita penyakit stroke….. juga buat kita2 yang sehat. Ya gak…..????

Sumber: NATURE CLINICAL PRACTICE NEUROLOGY, MAY 2008 VOL 4 NO 5

1. Methods for protein crystallization
2. Phase diagrams
   • Explanation of phase diagrams and crystallization
   • Movements in phase space for the different crystallization methods
3. Screening: searching phase space

Frequently Asked Questions about Crystals for Students contains an introduction to crystal growth (although it is written for small molecule crystallization).Douglas Instruments have a page on Experimental Design, particularly as it relates to automated screening, with phase diagrams and multivariate design.

Most of the work on the theory of protein crystallization has been done on lysozyme

• Tuhin Virmani’s pages show the theory of crystallization and phase diagrams for lysozyme and BSA
• NASA has an animated gif of lysozyme crystals growing in microgravity

Molecular Structure Corporation’s pages (MSC) have instructions for

• four different lysozyme crystallization procedures
• horse skeletal muscle myoglobin crystallization procedure

Vapour Diffusion Experiment

In a vapour diffusion experiment, small volumes of precipitant and protein mixed together and the drop equilibrated against a larger reservoir of solution containing precipitant or another dehydrating agent.

Phase diagram for vapour diffusion

Hanging Drop
Trays for hanging drop experiments VDX plates, greased or ungreased, from Hampton Linbro trays from Linbro Scientific, also from Hampton or Molecular Dimensions Costar trays, from Corning/Costar, also from Hampton Q-Plate and Q-Plate II from Hampton or Molecular Dimensions Coverslips All sizes and thicknesses and siliconized or unsiliconized from Hampton. All sizes and siliconized from Molecular Dimensions DIY siliconizing rack from Molecular Dimensions. Greasing Pregreased VDX plates from Hampton Silicone sealant from Hampton or Molecular Dimensions Grease Applicator from Molecular Dimensions

Sitting Drop
Trays for sitting drop experiments As for hanging drop with microbridges from Hampton MVD/24 Crystal Growth Chambers from Charles Supper Cryschem Plates from Hampton Crystal Clears from Douglas Instruments, also available from Hampton or Molecular Dimensions Coverslips or Tape Coverslips as above Sealing tape from Hampton or Molecular Dimensions

Sandwich Drop
Trays for sandwich drops Q-Plate from Hampton and Molecular Dimensions                                                                                                                Coverslips or Tape as above

Phase diagram for batch experiment

Trays for batch experiments 48 well trays from Hampton 96 well trays from Hampton or Corning/Costar For more information see Douglas Instrument’s Research Papers.

Phase diagram for dialysis experiment

Dialysis buttons are available from HamptonTrays for dialysis as for hanging dropGood Links Johan Zeelan’s notes on equilibrium dialysis Hampton’s Crystallization by Microdialysis

The solubility of proteins can be represented in phase diagrams.

The phase diagram plots the solubility curve of a protein.

• the horizonal axis shows the parameter being varied (usually precipitant concentration)
• the vertical axis shows the protein concentration.

Phase Diagram for a typical protein
 

• Saturation occurs when the rate of loss and gain of both the solid and solution phases of the protein are equal, and the system is in equilibrium.
• Salting-out is seen on the right hand side of the diagram where there is a reduction in protein solubility as the concentration of salt increases.
• Salting-in is seen on the left hand side of the diagram where there is an increase in protein solubility as the concentration of salt increases.

Energy Diagram for Crystallization

• The critical nucleus corresponds to the higher energy intermediate.
• The higher the energy barrier, the slower the rate of nucleation.

• the greater the likelihood that a critical nucleus will form
• the smaller the nucleus needed to induce crystal formation

This can be represented on a phase diagram by dividing the supersaturated zone into regions of increasing probability of nucleation and precipitation.

Phase Diagram showing zones for crystal nucleation, growth and precipitation.

Vapour Diffusion Experiment

In a vapour diffusion experiment where equal volumes of precipitant and protein are added in the drop, both the precipitant and protein concentration will double.

Phase Diagram for vapour diffusion experiment, no crystals

However, if crystals begin to grow, the concentration of protein in solution will decrease.

Phase Diagram for vapour diffusion experiment, crystals growing

In batch crystallization the precipitant and protein concentration stay the same.

Point A: Protein stays undersaturated
Point B: Protein crystallizes and the concentration of protein in solution drops to saturation
Point C: Protein precipitates, but crystals may still grow

Phase Diagram for batch experiments

In a salting-out experiment, the precipitant concentration increases.

Phase Diagram for a salting-out dialysis experiment

Dialysis has the advantage the the precipitant concentration can be altered during the course of the experiment.
You can also increase the concentration of one precipitating agent while decreasing the concentration of another.

Phase Diagram for a dialysis experiment, changing buffers

Dialysis can also be used to exploit the salting-in region of the phase diagram by forcing the protein out of solution by lowering the precipitant concentration.

Phase Diagram for desalting

Screening Methods

Full Factorial

In full factorial screens, all elements of the matrix of parameters are sampled

Incomplete Factorial

Incomplete factorial screening is a method of sampling parameter space evenly and efficiently.
Factor levels are chosen randomly and  then balanced to acheive uniform sampling.
All two-factor interactions are sampled as uniformly as possible.

Random

Purely random sampling of all parameters, but it approximates incomplete factorial designs.
 

Sparse Matrix

Sparse Matrix screens involve an intentional bias towards combinations of conditions that have worked previously.

Frequently Asked Questions about Crystals for Students

If have a question about crystals or crystal growing, look here first to see if your question has already been answered. Thanks goes to Alanna Windsor and Katie Boyle (no relation to me) as well as Carmella and April (from Maryland) for asking such good questions.

• What is a crystal?
• What types of crystals are there?
• How do crystals form and how do they grow?
• Why do different crystals have different shapes and sizes?
• What do you consider a perfect crystal?
• How does light affect the color of a crystal?

What is a crystal?

The definition I use in the course I teach on X-ray Crystallography is this:

“A crystal is homogenous solid exhibiting a high degree of internal order and a definite although not necessarily stoichiometric overall chemical composition.”

For simplicity, I usually only talk about systems which exhibit 3 dimensional order. There are, however, materials which display a high degree of internal order, but whose structures cannot be described by a 3 dimensional lattice.

By high degree of internal order means that the material is ordered over many atomic dimensions. Short range order means that order exists only a few atomic dimensions. For example, Silica (SiO₂) can exist as quartz (a crystal), or as glass (like in your windows, and has only short range order).

In addition, Tony Linden (alinden@oci.unizh.ch) adds:

A crystal can consist of any virtually pure single chemical compound (small impurities can be present and often give rise to colors in minerals, and mixtures of compounds can co-crystallize, but are less common). The compound may be inorganic, as in minerals and salts, e.g. SiO₂ (quartz) or NaCl (salt), or organic, such as sugar. In fact, just about any pure organic substance can be crystallized given the right conditions. Chemists actually use this process to purify their compounds as traces of impurity generally remain in solution when the crystals are formed. While classic compounds such as CuSO₄ can produce huge crystals and are the common classroom example because of this, most compounds crystallize as much smaller crystals and the solvents and techniques needed to obtain well formed crystals can vary from one compound to the next (these techniques are the primary focus of our web site). Even proteins can be encouraged to crystallize nicely under appropriate conditions and this is very important for scientists to be able to determine the structure of proteins and thereby understand their function. Protein crystallography is a very hot field in the biosciences these days.

What types of crystals are there?

There are a couple of ways to answer this question. I generally think in terms of crystal systems and lattice types. There are 7 crystal systems:

triclinic, monoclinic, orthorhombic, tetragonal, trigonal, hexagonal and cubic.

Lattices can either be primitive (only one lattice point per unit cell) or non-primitive (more than one lattice point per unit cell).

If you combine the 7 crystal systems with the 2 different types of lattices, you end up with 14 Bravais Lattices (named after Auguste Bravais who figured all this out in 1850).

Another way to answer your question is to catagorize crystals by their physical/chemical properties. In this classification you have four types of crystals:

Covalent Crystals: This is a crystal which has real chemical covalent between all of the atoms in the crystal. So really a single crystal of a covalent crystals is really just one big molecule. An example of this is a crystal like diamond or zinc sulfide. Covalent crystals can have extremely high melting points.

Metallic Crystals: Individual metal atoms sit on lattice sites while the outer electrons from these atoms are able to flow freely around the lattice. Metallic crystals normally have high melting points and densities.

Ionic Crystals: This is a crystal where the individual atoms don’t have covalent bonds between them, but are held together by electrostatic forces. An example of this type of crystal is sodium chloride (NaCl). Ionic crystals are hard and have relatively high melting points.

Molecular Crystals: This is a crystal where there are recognizable molecules in the structure and the crystal is held together by non-covalent interactions like van der Waals forces or hydrogen bonding. An example of this type of crystal would be sugar. Molecular crystals tend to be soft and have lower melting points.

Of course, this classification scheme has some ambiguous areas. For example, what of you have a crystal like [(CH₃)₂NH₂]⁺ (CH₃CO₂)^- is that a molecular crystal or an ionic crystal?

How do crystals form and how do they grow?

Crystals start growing be a process called “nucleation”. Nucleation can either start with the molecules themselves (we’ll call this unassisted nucleation), or with the help of some solid matter already in the solution (we’ll call this assisted nucleation). I’ll write about both. Before I do that, here is a general explanation from Tony Linden:

Once a solution is saturated, or a melt nears the solidification point, solid material starts to form. If the molecules come together in a random arrangement, they do not occupy the closest packed space. However, if the molecules come together in an ordered array, they pack together in a much smaller space, like in a properly assembled jigsaw puzzle (see how much more space an unassembled puzzle occupies when the pieces are randomly positioned to touch each other, but not overlap). Thus, the proper packing uses less space and is also of lowest energy, which is always the most stable condition. As it happens, this ordered array pattern repeats itself regularly in 3 dimensions, and the crystal is the macroscopic object we see as a result. The nice faces of a crystal result from the fact that certain directions in this array are more accessible to the attachment of new molecules, so the crystals grow uniformly in these directions. However, the incoming molecules need a little time to align themselves properly at the surface of a growing crystal in order for the crystal to continue to grow nicely. Hence the need for slow crystallization. If the solution becomes over-saturated so that solid forms quickly, the incoming molecules do not have time to align properly with the result that one obtains small crystals that are usually poorly formed.

Now we can talk a bit about nucleation, a very important step in crystallization.

Unassisted nucleation:

When molecules of the “solute” (the stuff of which you want to grow crystals) are in solution, most of the time they see only solvent molecules around them. However, occaisionally they see other solute molecules. If the compound is a solid when it is pure, there will be some attractive force between these solute molecules. Most of the time when these solute molecules meet they will stay together for a little while, but then other forces eventually pull them apart. Sometimes though, the two molecules stay together long enough to meet up with a third, and then a fourth (and fifth, etc.) solute molecule. Most of the time when there are just a few molecules joined together, they break apart. However, once there becomes a certain number of solute molecules, a so-called “critical size” where the combined attractive forces between the solute molecules become stronger than the other forces in the solution which tend to disrupt the formation of these “aggregates”. This when this “protocrystal” (a sort of pre-crystal) becomes a nucleation site. As this protocrystal floats around in solution, it encounters other solute molecules. These solute molecules feel the attractive force of the protocrystal and join in. That’s how the crystal begins to grow. It continues growing until eventually, it can no longer remain “dissolved” in the solution and it falls out (as chemists like to say) of solution. Now other solute molcules begin growing on the surface of the crystal and it keeps on getting bigger until there is an equilibrium reached between the solute molecules in the crystal and those still dissolved in the solvent.

Assisted Nucleation:

Pretty much the same thing happens as in unassisted nucleation, except that a solid surface (like a stone, or brick) acts as a place for solute molecules to meet. A solute molecule encounters the surface of a stone, it adsorbs to this surface, and stays on it for a certain time before other randomizing forces of the solution knock it off. Solute molecules will tend to adsorb and aggregate on the surface. This is where the protocrystal forms, and the same process as described above happens.

You can probably see from what I wrote above, why solution in which the concentration is near saturation, that crystals grow fastest. If there are more solute molecules in a given volume, then there is more of a chance they will meet one another. You also don’t want to heat up the solution because that acts as the major randomizing force in solution which causes the aggregates of molecules to break up.

Why do different crystals have different shapes and sizes?

This depends on 2 factors: 1) The internal symmetry of the crystal, and 2) The relative growth rates along the various directions in the of the crystal. For example, suppose you have mutally perpendicular axes, a, b, and c. Suppose the crystal grows at equal rates along a, b, and c, then the crystal shape will be a cube. Now suppose a different crystal grows fast in the a and b direction, but very slowly in the c direction. The crystal will then grow as thin plates with the face of the plate being perpendicular to c. These are only simple examples. More complicated cases (and shapes) happen when the crystal doesn’t have mutually perpendicular axes, and when the fastest directions of growth correspond to face or body diagonals (or even other directions) in the crystal.

Why crystals grow at different rates in different directions is a very complicated question. If there is a highly attractive interaction (energetically speaking) along a certain direction of a crystal, then that direction will probably grow fast. However, it could also grow slowly, if that direction interacted strongly with the solvent; having strongly absorbed solvent on the surface of the crystal could block growth along that face.

What do you consider a perfect crystal?

A perfect crystal is one which has a single lattice (i.e. not a twinned crystal) and is completely regular, free of defects and dislocations. Most single crystals, however, are imperfect in the sense that they are composed on regions of slight relative misalignment (about 0.1 to 0.2 degrees). For X-ray diffraction (which is what I do), this slight imperfection is desirable because the diffracted intensities from such a crystal are higher than those whose lattices are “perfect”. When a crystal is too perfect, the X-ray diffraction pattern suffers from what is called “extinction”. Crystallographers then talk about an “ideally perfect” and “ideally imperfect” crystals. We use these terms to talk about real crystals which behave in accordance with simple theories of X-ray diffraction.

How does light affect the color of a crystal?

The color of any compound (whether or not it is a crystal) depends on how the atoms and or molecules absorb light. Normally white light (what comes out of light bulbs) is considered to have all wavelengths (colors) of light in it. If you pass a white light through a colored compound some of the light is absorbed (we don’t see the color which is absorbed, but we see the rest of the light) as it is reflected off the surface. This gives rise to the idea of “Complementary Colors”. If a compound absorbs light of a certain color the compound appears to be the complimentary color. Here is a table of colors and thier compliments:

So if you have a crystal which absorbs red light, it will appear green. Conversely, if the crystal absorbs green light, it will appear red.

With regard to the optical properties of crystals, Tony Linden, adds:

One interesting feature of some crystals is their effect on an image viewed through them. A calcite crystal placed over a cross on a page will make the cross look doubled because of total internal reflection within the crystal.

Pembahasan ini diambil dari situs: http://www-structmed.cimr.cam.ac.uk/Course/Crystals/theory.html

Mungkin ada temen2 yang ingin tahu prinsip2 dan metode yang dilakukan dalam penelitianku, silahkan baca artikel beriku ini…

Overview of macromolecular X-ray crystallography

Outline

• Methods to study 3D structure
• Principles of X-ray crystallography
• Fitting and refinement
• Validation

This series will cover the theory and practice of X-ray crystallography, particularly as it applies to studying the three-dimensional structure of macromolecules. The notes will often refer specifically to proteins, but generally the same techniques and problems apply to other macromolecules (DNA, RNA) or assemblies (viruses, ribosomes).

If you’ve gotten this far you probably have a good idea why we want to study the 3D structure of molecules. 3D structure allows us to understand biological processes at the most basic level: which molecules interact, how they interact, how enzymes catalyse reactions, how drugs act. In some cases, it can allow us to understand disease at an atomic level, such as the sickling of red blood cells. We can also exploit 3D structure in developing new drugs.

There are a number of methods for studying 3D structure, outlined briefly in the next section. But we will concentrate on X-ray crystallography, which is arguably the most effective of these techniques at present. Certainly the other techniques complement crystallography and have a valued place in the set of tools that we use.

Methods to study 3D structure

Crystallography

If you think about how you determine the shape of objects around you, the most obvious is just to look at them. If they’re small, you use a microscope. But there’s a limit to how small an object can be seen under a light microscope. The limit (the “diffraction limit”) is that you can not image things that are much smaller than the wavelength of the light you are using. The wavelength for visible light is measured in hundreds of nanometers, while atoms are separated by distances of the order of 0.1nm, or 1Å. Looking at the electromagnetic spectrum, X-rays get us in the right wavelength range.

But we can’t build an X-ray microscope to look at molecules, for two reasons. First, we don’t have an X-ray lens. Second, even if we did have one, it would have to be made with tolerances significantly less than the distance between two atoms! However, we can (in effect) simulate an X-ray lens on a computer. You can think of a microscope as working in two stages. First, light strikes the object and is diffracted in various directions. The lens collects the diffracted rays and reassembles them to form an image. With X-rays, we can detect diffraction from molecules, but we have to use a computer to reassemble the image. It’s not quite so simple, but that’s the essence of the method.

We can consider other types of waves with wavelengths in the correct range. One of the wonderful and unexpected results of quantum mechanics is that particles have a wave nature. The faster they are moving, the shorter the wavelength. Two types of particles can be accelerated to speeds sufficient to bring their wavelengths into the Ångstrom range: neutrons and electrons. Neutron diffraction works more or less like X-ray diffraction.

Electron microscopy

Electrons diffract too, but they can also be focused by magnetic fields, which allows the construction of electron microscopes. The very best electron microscopes have resolving powers near atomic resolution. There are special problems with sample damage, which means that very low electron doses must be used to avoid destroying the sample, so that the images have extremely poor signal-to-noise and must be averaged. It turns out that electron microscopy tends to be most useful for very large assemblies, which is where crystallography tends to become very difficult, so that the techniques are quite complementary. But there won’t be space or time to go into all of that here.

Atomic force microscopy

Apart from looking at things to determine their shape, you can also use your sense of touch. That, in essence, is what a number of techniques based on scanning microprobes do.

Nuclear magnetic resonance

Here we stray from analogy with our senses. NMR uses much more indirect methods to determine 3D structure. It is based on the quantum mechanical properties of atoms, particularly spin, and it determines information about atoms from the fact that their local environment influences how they respond to applied magnetic fields. The kind of information that can be obtained includes the measurement of interatomic distances, and features of the spectrum (coupling constants) that can be interpreted in terms of torsion angles.

Principles of X-ray crystallography

Why X-rays?

As noted above, the use of electromagnetic radiation to visualise objects requires the radiation to have a wavelength comparable to the smallest features that you wish to resolve. We often use X-rays emitted from copper targets bombarded with high energy electrons, which emit at several characteristic wavelengths: the one we use is called CuKα, which has a wavelength of 1.5418Å. This is very similar to the distance between bonded carbon atoms, so it is well suited to the study of molecular structure.

Why electron density?

What we see as the result of a crystallographic experiment is not really a picture of the atoms, but a map of the distribution of electrons in the molecule, i.e. an electron density map. However, since the electrons are mostly tightly localised around the nuclei, the electron density map gives us a pretty good picture of the molecule.

This is because electromagnetic radiation (including X-rays) interacts with matter through its fluctuating electric field, which accelerates charged particles. You can think of the electrons fluctuating in position and, through their accelerations, emitting electromagnetic radiation in turn. Because electrons have a much higher charge to mass ratio than atomic nuclei or even protons, they are much more efficient in this process. Intensity of scattered radiation is proportional to the square of the charge/mass ratio, and the proton is about 2000 times as massive as the electron.

Why crystals?

X-ray scattering from a single molecule would be unimaginably weak and could never be detected above the noise level, which would include scattering from air and water. A crystal arranges huge numbers of molecules in the same orientation, so that scattered waves can add up in phase and raise the signal to a measurable level. In a sense, a crystal acts as an amplifier.

Of course, if the waves add up in phase in some directions, they have to cancel out in a lot of other directions. That is why the diffraction pattern from a crystal is an array of spots.

There are a number of potential bottlenecks in determining a crystal structure, but growing a useful crystal can be the most serious one. If you can’t collect any data or only bad data, you won’t be able to solve a structure. So we’ll spend the next lecture looking at the theory and practice of crystallisation.

Diffraction

Think of the electrons in a crystal as emitters of little waves. When the little waves emit add up, they interfere with one another: they can add up in phase, out of phase, or something in between. Which they do depends on the direction of the incoming and outgoing waves and the positions of the electrons relative to each other. The total path from the source to the detector will determine what happens. If the difference in path taken via one electron and another is a multiple of the wavelength, then the waves will scatter in phase and their amplitudes will add up. If it’s a half-integral multiple of the pathlength, they will scatter exactly out of phase and cancel out.

The conditions for scattering in phase can be summed up quite neatly if you think of the waves being reflected off of planes passing through the atoms. The relationship between scattering angle and the interplanar spacing is given by Bragg’s law, which we will go into analytically at a later date. For the meantime, there is a Bragg’s law applet that allows you to play with wavelength, interplanar spacing and angle of incidence and reflection. Notice that, if you increase the wavelength, the total diffracted intensity becomes less sensitive to the spacing or to changes in angle. That means that the diffraction pattern becomes less sensitive to the fine details. Also notice that, if you keep the wavelength fixed but decrease the spacing, you have to go to higher angles to get the first peak in diffracted intensity. Because of this inverse relationship between the spacing in the object and the angle of diffraction, the diffraction space is usually called “reciprocal space”. The further out you go in reciprocal space, the more the diffraction pattern becomes sensitive to objects that are close together in “real space”. We’ll spend a lot of time later dealing with the properties of reciprocal space and how to think about it.

One more thing to notice: the position of the peaks in the wave that results from the interference of scattering from two atoms depends on their relative position. The position of the peak in a wave is described by its phase.

Fourier theory

It turns out (for reasons that we’ll go into in great depth in the advanced series) that the diffraction pattern is related to the object diffracting the waves through a mathematical operation called the Fourier transform. If you think of the electron density as a mathematical function, then the diffraction pattern is the Fourier transform of that function.

This means that, for a real understanding of crystallography, you need to understand some properties of Fourier transforms, and we’ll spend some time on that later. But for now we’ll content ourselves with one property of Fourier transforms: they can be inverted. If you apply a Fourier transform to some function, you can take the result and run it through an inverse Fourier transform to get the original function back. (The inverse Fourier transform is essentially just another Fourier transform.) This is how we can use a computer to take the diffraction pattern and give us a picture of the electron density.

But there’s a problem, and it’s such an important problem that it’s usually referred to in capital letters as The Phase Problem. The Phase Problem arises because we need to know both the amplitude and the phase of the diffracted waves to compute the inverse Fourier transform. But what we measure in the experiment is essentially a count of the number of X-ray photons in each spot. We have no practical way of measuring the relative phase angles for the different diffracted spots experimentally. The number of photons gives the intensity, which turns out to be proportional to the square of the amplitude (peak height) of the diffracted wave. But the phase has been lost.

The phase, in fact, is extremely important, as you can see from taking a look at Kevin Cowtan’s Book of Fourier.

Kevin has another useful web page, the Interactive Structure Factor Tutorial, which provides a nice tool to get a feel for how the inverse Fourier transform works.

The Phase Problem

Apart from growing useful crystals, this is often the most serious bottleneck in determining a new structure. Because we can’t measure the phase directly, we have to deduce it indirectly. We’ll go into this in more detail later, but there are basically two approaches:

Perturb the structure and diffraction pattern

The classic technique along these lines is isomorphous replacement. Isomorphous means “same shape” and, essentially what you do is to get a crystal that is nearly identical to the one you’re studying, except that a few atoms have been replaced or added. If these atoms are “heavy”, i.e. they have a large atomic number, they will perturb the diffraction pattern. It is possible to deduce the positions of the few heavy atoms and from that to deduce possible values for the phase angles.

A conceptually similar technique uses just one crystal, which contains atoms called anomalous scatterers. By changing the wavelength of the X-rays, you can change the degree to which the anomalous scatterers perturb the diffraction pattern, which gives the same kind of information as isomorphous replacement. This technique, called multiple-wavelength anomalous dispersion (MAD) will be covered in its own lecture in the advanced series.

Guess the phases

If you have a good idea of what the structure should look like (you’ve seen it in another crystal form, or you know the structure of a closely-related protein) you can place a model in the crystal and compute guesses for the phases. This technique is called molecular replacement, and we’ll go into great detail about this too later.

Resolution

If the atoms were completely still, the molecules throughout the crystal were in identical conformations, and the crystal were perfectly ordered, then all the molecules would scatter in phase regardless of the angle of scattering and we would be able to collect diffraction data to a limit imposed only by the wavelength of the X-rays. The electron density map would have peaks at each of the atomic positions. But reality is rarely so favourable. Proteins are generally fairly flexible, and crystals have lattice disorder, i.e. the repeating units are not necessarily perfectly aligned throughout the crystal. So as we start to look at finer details by going to higher scattering angles, the diffraction pattern starts to cancel out. For this reason, most protein structures are limited to a level of detail where atoms are not resolved from one another. What we see is typically tubes of electron density for atoms that are bonded together.

Examples of maps at different levels of resolution can be seen in Phil Evans’ page on fitting and refinement.

Fitting and refinement

Because the density map doesn’t resolve individual atoms, fitting models to density is a bit of an art. It requires the use of computer graphics programs such as coot or O. Early in a structure determination, the phase information is usually poor as well, so the electron density maps are not ideal. As a result, the initial model that one builds will have a lot of errors.

An atomic model can never be perfect, but it can be improved a great deal by a process called refinement, in which the atomic model is adjusted to improve the agreement with the measured diffraction data. We’ll look at this in an overview, and eventually in greater sophistication when we consider the use of maximum likelihood to improve models.

The success of an atomic model is often judged through the standard crystallographic R-factor, which is simply the average fractional error in the calculated amplitude compared to the observed amplitude. Though it depends on a number of factors, as a rule of thumb a good structure will have an R-factor in the range of 15% to 25%.

Validation

It turns out that, for most protein structures, there is a very poor observation-to-parameter ratio. For each atom there are 3 or 4 parameters (3 describing its position, possibly one indicating how mobile it is). At a typical resolution, there will only be about one observation for each parameter. Because there are not enough data, we typically supplement the diffraction data with restraints on geometry, which keep the bondlengths, angles and close contacts in a reasonable range.

With enough parameters you can fit an elephant, so it is easy to overfit the data and get a misleading level of agreement with the observed data. A valuable way to detect this is to leave out a fraction of the data from use in refinement. These cross-validation data should be free of the effects of overfitting, and they allow you to compute an R-free, which is an unbiased indication of the quality of the structure.

Other tools are used for validation. What is often felt to be most useful for validation is something that isn’t used explicitly in the refinement process. For instance, the main-chain torsion angles are hard to restrain in refinement, but the distribution of these angles in the Ramachandran plot is very restricted. The Ramachandran plot is thus a good indicator of the quality of a structure. Other indicators are used, such as the distribution of hydrophobic and hydrophilic amino acids. To see a full-sized Ramachandran plot, click on the thumbnail below.

Many validation tools are available, and many can be run interactively on the Web. For instance, you can upload a PDB file containing the atomic coordinates for a protein to the Validation Suite at the EBI. This will run a whole series of tests, and present the results to you in tables and graphs. A newer alternative is the MolProbity server, provided by Jane and David Richardson at Duke University.

Diambil dari: http://www-structmed.cimr.cam.ac.uk/Course/Overview/Overview.html

Sebenarnya, selama dua minggu sejak penelitian dimulai, banyak ilmu yang aku peroleh dan juga sangat menyenangkan. Selain karena emang bidang yang dilakukan sangat baru, co-supervisorku juga ternyata orang Indonesia yang dapat menjelaskan setiap langkah dengan jelas dan mudah dipahami.Ya, cuma masalahnya, karena di labku ada tiga orang Indonesia, jadi tiap hari ngomongnya pake bahasa Indonesia terus. Jadinya Bahasa Inggrisku tidak ada peningkatan sama sekali. Hehehe….

Oke, sekarang kita bahas mengenai judul cerita ini. Aku hari ini boring sekali karena emang gak ada kerjaan. Jadi, sekarang itu aku lagi nunggu kristalisasi protein. Ya, gak ada yang dilakukan, selain berdoa semoga kristalnya tumbuh tentunya. Ya begitulah… dari pagi, aku ikut pertemuan mingguan segroup kristalografi, trus ngeliatin co-supervisorku kerja. Tadinya aku mau diskusi sama profesorku. Tapi, pas keruangannya, ternyata dia lagi sibuk. Katanya lagi ngoreksi papernya salah satu mahasiswanya. Padahal kita teh udah janji pas hari jum’atnya. Tapi gak apa2 deh, rencananya diskusinya diubah jadi besok pagi. Akhirnya aku pun ke ruanganku lagi dan browsing2 gak jelas. Dan, ngantuk deh…. BAAAAAAAAANGEEEEEEEETTT….. Mau pulang, tapi harus nunggu jam lima. Soalnya, di sini, kerja dimulai dari jam sembilan sampai jam lima sore. Jadi, karena saat itu baru jam setengah tiga sore, terpaksa harus bengong dulu selama dua setengah jam untuk pulang. Boring banget kan…..??????

Tapi, semoga saja kristalnya tumbuh…… Doain ya. Pas tadi pagi dicek, belum tumbuh sih, cuma udah mulai terjadi presipitasi. Kata co-supervisorku, presipitasi itu menunjukkan telah terjadi tahap supersaturasi. Maksudnya si-proteinnya sudah sangat jenuh karena pelarutnya jumlahnya sangat sedikit. Semoga saja besok udah mulai tanda2 tumbuhnya kristal…. Amin.

O ya…. buat pembaca yang tetep dengan setia menanti kelanjutan “awal mula….”, maaf ya, ceritanya belum dilanjutkan lagi. Soalnya nunggu saat yang tepat supaya moodnya pulih. Sekalian cari inspirasi agar ceritanya menarik. Sebenarnya pasti menarik sih, soalnya klimaksnya di sana. Jadi, perjalanan ke Jakarta yang heboh itu baru awal dari masalah2 besar lainnya. Tunggu ya……. Hehehehe…..

Jam setengah dua belas malam aku pun mulai siap-siap. Pasport, foto kopi pasport, foto-foto, semuanya dah siap. Aku pun meninggalkan kosan untuk pergi ke pool X-Trans. Karena jaraknya lumayan dekat, sekitar  satu kiloan lah, aku jalan kaki ke sananya. Lagian kalo ngangkot harus dua kali (naik angkot caheum-ledeng, dan chaheum-ciroyom). Males ah, mendingan jalan kaki.

Jalanan udah mulai sepi dan gelap. Di beberapa tempat ada pedagang kaki lima yang sudah mulai sepi pelanggan dan bersiap-siap untuk kembali ke peraduannya. Kira-kira lima belas menit kemudian, aku pun sampai di pool X-Trans. Walaupun buka 24 jam, tapi jam segitu, pool bener-bener sepi. Di sana cuma ada beberapa orang yang sedang jaga. Sebenarnya aku juga agak heran, kenapa sekitar 15 menit lagi akan berangkat (saat itu jam 00.15), kok penumpang lain belum ada yang dateng. Ah, mungkin sebentar lagi. Pikirku. Akhirnya aku pun masuk ke dalam dan menghampiri meja tempat pembelian tiket. Di sana ada seorang lelaki yang sedang duduk-duduk sambil menonton siaran tv kabel. 

“Mas, mau beli tiket ke Blora untuk jam setengah satu.” Kataku. Beberapa saat lamanya lelaki itu hanya diam sambil memandangiku.

“Setengah satu siang?” Tanyanya.

“Bukan, sekarang.” Jawabku. Mendengar jawabanku, keliatannya dia tambah bingung. Tapi aku gak curiga apa2 soalnya tadi kan aku dah booking tiketnya.

“Tapi, untuk keberangkatan jam setengah satu pagi nggak ada mas.” Katanya.

Deg…… Aku kaget sekaligus heran. “Tapi, tadi saya udah pesen tiketnya”

“Ah, salah mungkin mas. Soalnya paling pagi, travel ini berangkatnya jam setengah dua. Itu pun bukan ke Blora, tapi ke Kartika Chandra.”

“Enggak-Enggak. Gak mungkin salah. Tadi saya sendiri kok yang pesen.” Kataku dengan nada agak sedikit meninggi.

“Tapi, untuk keberangkatan jam segitu gak ada. Tuh coba liat daftarnya” Kata dia sambil ngeliatin daftar dan jadwal keberangkatan travel. Trus dia nunjukkin leaflet Travel yang ada jadwal keberangkatan dan tujuannya.  ”Nih, coba liat mas, untuk ke Blora, gak ada yang jadwalnya jam setengah satu pagi. Paling pagi juga jam empat. Tapi itu pun sekarang udah penuh.”

Mendengar kata-kata lelaki itu, hatiku makin panas. “Enggak ah…. Aku tadi pesen sendiri ke sini. Pesen tiket ke Blora untuk keberangkatan jam setengah satu pagi. Beneran…. Aku sendiri malah ngeliat si mbak yang di bagian pemesanannya nulis nama sama no HP ku.” Aku gak mau kalah.

“Emang tadi yang nerimanya siapa namanya?” Tanyanya.

“Nggak tau. Pokoknya sama cewek2 di sana” Jawabku sambil nunjuk ke tempat pemesanan tiket. Trus aku ceritain apa yang terjadi tadi siang.

“Makanya mas, nanti kalo pesen tiket lagi, tanya nama orang yang nerimanya siapa. Jadi kalo ada kesalahan bisa tau siapa orangnya.” Jawabnya cuek sambil ngebolak balik daftar pemesanan.

Yeeeeee….. Ngapain nanya2 namanya segala. Apa urusanku. Pikirku dalam hati. Aku kan gak nyangka akan kejadian hal seperti ini. Lelaki itu pun mencek namaku ke semua berkas pemesanan ke semua jurusan yang ada, ke semua waktu. Siapa tau tadi salah ngisiin. Tapi ternyata namaku gak ada di satu pun berkas itu. Namaku hilang, bagai di telan bumi. Aneh…..

Mengalami hal seperti ini, aku bingung tujuh keliling. Gimana ke Jakartanya? Tapi saat itu aku belum memberi tau An mengenai masalah ini. Soalnya aku yakin dia lagi di jalan menuju ke sini. Nanti dia kaget kalo aku kasih tau sekarang. Aku biarin aja dia nyampe dulu di sini. Biar dia kagetnya di sini.

Akhirnya setelah panjang lebar berdebat dengan lelaki itu, dia nyaranin supaya aku duduk dulu dan dia mau coba cari lagi berkas pemesanannya. Aku pun duduk di kursi depan dengan pikiran kusut. Bingung. Bagai mana bisa namaku gak ada diberkas. Lelaki itu juga keliatan bingung dan terus mencari-cari namaku. Dia kadang-kadang nelepon sana-sini. Apa yang ada di pikiranku saat itu adalah “Bagaimana pun caranya, aku harus berangkat ke Jakarta dengan Travel ini sepagi mungkin”. Bagaimana tidak, aku ngerasa dipermainkan. Jadi pihak travel harus bertanggung jawab dong.

Tak berapa lama kemudian An datang. Aku pun segera menceritakan apa yang terjadi. Ya… pasti dia juga kaget mendengar apa yang terjadi. Kemudian dia nelpon “nggak tau siapa”. Yang pasti, setelah itu dia nawarin untuk naik kereta api. Tapi aku berpendapat untuk tetap bertahan di sini. Aku ingin tau apa yang terjadi sebenarnya. Dimana berkas yang tertulis namaku? Hanya itu. Kalo emang ternyata salah tulis, gak apa2. Yang penting aku tau dimana namaku ditulis. Abis itu baru memikirkan langkah selanjutnya. Pokoknya aku harus tau dimana si Mbak yang tadi siang menulis namaku.

Karena jam udah menunjukkan jam satu pagi lebih, aku pun menghampiri lelaki itu lagi.

“Gimana mas?” Tanyaku.

“Sebentar…. Nama mas gak ada di daftar pemesanan mana pun. Mungkin nama Mas dihapus.” Loh, kok? pikirku.

“Tapi, tenang dulu mas. Saya mau nelepon ke calon penumpang yang mau berangkat jam setengah dua ke Kartika Chandra. Siapa tau ada yang membatalkan pemesanannya”.

Akhirnya aku pun kembali ke kursi depan sambil nunggu berita dari lelaki itu. Tak berapa lama kemudian lelaki itu bilang ada kursi yang kosong dua buah untuk keberangkatan ke Kartika Chandra. Ya, boleh deh. Kemana pun, asal ke Jakarta. Dari sana ke kedutaan mah urusan nanti. Pikirku. Syukurlah…… 

Akhirnya jam setengah dua pagi, kami pun berangkat ke Jakarta dengan tujuan ke Hotel Kartika Chandra. Karena dari Bandung jam setengah dua pagi, maka sampai sana jam setengah empat pagi. Masih pagi banget kan. Jadinya, kami nunggu di pool X-Trans sampai pagi. Kebetulan di sana ada TV, jadi kami bisa nunggu pagi sambil nonton.