Crystallography: phasing out heavy atom derivatives

REFERENCE: Liu et al. "Structures from Anomalous Diffraction of Native Biological Macromolecules." Science (2012) 336, pgs 1033 - 1037.                 

        The field of protein crystallography has partially pulled back the veil that lies between the macroscopic and microscopic worlds.  Coupling crystallography with X-ray diffraction yields structure models that allow scientists to consider the shape, surface, and, in short, the angstrom-length details of a protein.  Each spot on a diffraction pattern arises from a constructive interference event between X-ray waves within the protein crystal.  Amplitude and frequency information, two of the three variables that define a wave, are provided by these spots, but the final parameter of phase must be experimentally determined before a protein model can be built.  “Solving the phase problem” has thus far relied on soaking proteins in heavy atoms, use of experimental phases from a closely related protein, or incorporation of selenomethionine into the polypeptide chain.  Scientists would rather determine structures using crystals of native protein but such a method has thus far been unavailable.  In a recent study published in the journal Science, Liu et al. discuss their new technique that uses anomalous dispersion from native atoms within the protein to obtain phase information; the authors further support their technique’s worth by solving the structures of four different proteins that vary in size and subcellular localization.

                Anomalous dispersion relies on a heavy atom’s ability to absorb and emit X-rays.  When the wavelength of the X-rays approaches the characteristic emission wavelength of the atom, then the absorption drops off sharply giving rise to anomalous scattering.  With access to tunable synchrotron beamlines and protein crystals bearing heavy atoms, crystallographers using multiple anomalous dispersion (MAD) or single anomalous dispersion (SAD) can gather data sets at their heavy atom’s absorption edge to identify the heavy atoms’ locations and eventually provide the phases necessary to build a protein model.  Selenium (Z = 34) has traditionally been added to proteins to create anomalous signals, but obtaining phase information from crystals of native protein would be optimal. Intrinsic iron (Z = 26) has occasionally been sufficient for phase determination, but the next heaviest atom regularly found in proteins is sulfur (Z = 16).  .  

                Since 1981, 57 novel structures have been published that employed light atom (Z <20) SAD to determine phases.  In comparison, over 5000 structures have been deposited into the Protein Data Bank (PDB, www.pdb.org) in the past fifteen years that used heavy atom SAD.  This discrepancy arises from the low anomalous strength of light atoms, low amount of sulfurs per protein molecule, radiation damage, and diffuse scattering of the X-rays.

                Determined to overcome these complications, Liu et al. began with their previously reported technique that improved data from poorly diffracting selenomethione protein crystals.  To decrease radiation damage and increase signal to noise, a procedure was designed to merge data from multiple crystals.  Building on this, authors then optimized the X-ray beam energy based on knowledge that anomalous signal from light atoms increases with increasing wavelength.  Together, these two methods increased signal to noise and minimized radiation damage.  Finally, the crystal and beam path were placed inside a helium-filled cone and the beam size was matched to crystal size, which also boosted signal to noise and reduced incoherent X-ray scattering.

                Four test proteins were crystallized: netrin G2, TorT/TorS_s, HK9s and CysZ.  Three of the four contained at least 20 sulfur atoms, but HK9s only had three plus an additional chloride.  At least 5 crystals were used per protein.  The authors then define three criteria each crystal’s data set must meet before being merged with others: unit cell parameters less than 3σ, overall diffraction dissimilarity less than 5%, and the relative anomalous correlation coefficient greater than 35%.  This is an improved rubric from their previous work.  Of the 31 crystals screened, only one data set was removed after failing the above standards.  For each protein, data sets were added together one at a time and tested for structure determination.  With each successive data set, anomalous signal, resolution, and electron density maps improved, thus validating their procedures.

                As a way to demonstrate the versatility of light atom anomalous dispersion, the test proteins were diverse.  They ranged from127 to 1148 ordered residues, their crystal symmetries varied from monoclinic to tetragonal and resolutions are as high as 2.3 Å.  Netrin G2 and TorT/TorS_s were both previously unknown structures, while HK9s and netrin G2 were not amenable to previous structure determinations.  CysZ is a membrane protein, while the rest of soluble domains of membrane proteins.  Finally, TorT/TorS_s is reported to be at a complexity level exceeding 90% of the current PDB and its structure was determined here to reasonable resolution.  The authors do admit that the technique will benefit from improvements at beamlines, scaling and weighting procedures, but they do believe that multicrystal SAD phasing will be extremely useful for determining de novo structures of native proteins and nucleic acids.

Influenza Research Pause

REFERENCE: Fouchier et al. “Pause on avian flu transmission studies.”  Nature (2012)

LINK directly to published letter

                As I discussed in my American Society for Cell Biology Meeting post, I don’t want to repeat work that has already been discussed outside of the initial scientific publication, however this topic is interesting, especially considering the letter was signed by 39 authors and published in both Nature and Science magazines.  

                Work being performed at the University of Wisconsin-Madison and Erasmus MC in the Netherlands has suspended important research on a highly transmittable influenza virus due to fears of viral escape from their laboratories.  They have imposed a 60 day “pause” on their work while the scientific community and the community at large have time to discuss some of the new issues this type of research presents.

American Society for Cell Biology Meeting

Denver, CO: December 3^rd (Saturday) – December 7^th (Wednesday)

REFERENCE:  Science (2011) 334 pgs 1046 - 1051

                This Saturday marks the beginning of the annual ASCB meeting in Denver, CO.  The event will feature over 3000 scientific poster presentations as well as 100 scientific sessions.  Principal Investigators, post doctoral associates and graduate students from all over the world will descend on Denver to discuss science (and ski) starting this weekend.

                As a preview to this event, Science magazine published a five article series discussing some of the most pressing questions currently facing cell biologists.  They include…

Do lipid rafts exist?  This is a contentious topic, but important to understand.  The plasma membrane is the first line of defense for a cell and acts as a gate keeper to all the comings and goings.  Understanding how it works is essential.

How does a cell know its size?  Many different kinds of cells exist but all of them stay within a certain size.  Certain proteins have been identified in yeast and bacteria that are involved in cells “sensing” their size but more work needs to be done.

How does a cell position its proteins?  Some cells make upwards of 10,000 proteins.  Positioning all of them so that they are in the proper places to perform their functions efficiently is a monumental task.  Proteins carry targeting sequences that place them in different organelles, but new research suggests that mRNAs may also be playing a role.

How do hungry cells start eating themselves? Autophagy is becoming a hot topic!

Does a gene’s location in the nucleus matter? Nuclear organization is important to cell function.  In fact, in cancer and other diseases, the nucleus is reorganized.  Researchers are trying to understand why the cell likes its proteins and RNA in certain places relative to its chromosomes.

I highly recommend reading them.  The articles are short and already written in a summary format.  I don’t want to write a redux of a redux on this blog because that is ridiculous.  Instead, I implore you to pick up a November 25^th copy of Science magazine and read pages 1046 – 1051!

Ancient Humans

REFERENCE

Rasmussen et al. “An Aboriginal Australian Genome Reveals Separate Human Dispersals into Asia.” Science (2011) 334 pgs 94 – 98. 

Two theories exist to explain the ancestry of Aboriginal Australians (Figure 5.1).  The first, called the Single-Dispersal model, claims that Africans split from Eurasians, which then became Europeans and Asians, which led to Aboriginal Australians.  Unfortunately, the split between Europeans and Asians is believed to have occurred 17,000 to 43,000 years ago but archeological data suggests that anatomically correct humans were in Australian around 50,000 years ago.

                The second model, called the Multi-Dispersal model, suggests that an earlier and perhaps independent dispersal occurred before the split between Africans and Eurasians.  

                To determine which model is correct, Rasmussen et al. sequenced the genomic and mitochondrial DNA from the hair of an early 20^th century Aboriginal male and detailed their findings in the most recent issue of Science.    They found that Aboriginal Australians shared significantly more derived alleles with Asians (Cambodian, Japanese, Han and Dai) than Europeans (French) and that Europeans shared more derived alleles with Asians than Aboriginal Australians. 

                The authors went on to sequence three Han Chinese genomes and used this data to support their conclusions that Aboriginal Australians split from African populations before Eurasians differentiated into Europeans and Asians, thus supporting a Mutliple-Dispersonal model.  Fitting well with archeological data, it was concluded that this split occurred 62,000 to 75,000 years ago while the European/Asian split was 25,000 to 38,000 years ago.

                Rasmussen et al. concede that making one Aboriginal Australian DNA sample representative of an entire population may not be entirely fair.  However, if true, Aboriginal Australians are the direct descendants of the first humans in Australia and “…likely have one of the oldest continuous population histories outside of sub-Saharan Africa today.”