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Acta Cryst. (2014). A70, C122
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The transmembrane subunit (gp41) of the envelope glycoprotein (Env) of HIV-1 associates non-covalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 is involved in many essential biological functions and its primary role is to anchor the Env in both viral and cellular membranes. Despite having many important biological functions, the atomic structure of gp41 TM domain remains unknown. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the prefusion or post-fusion conformations, which could not be recognized by the broadly neutralizing antibodies 2F5 and 4E10. Here we describe the expression, purification, biophysical characterization and crystallization of a chimera construct including maltose binding protein (MBP) and MPR-TM of gp41. The purified MBP-MPR-TM protein reacts with the broadly neutralizing antibodies 2F5 and 4E10 with nanomolar affinities and thereby may represent an immunologically relevant conformation mimicking a pre-hairpin intermediate of gp41. Crystals could not be obtained initially when MPR-TM was fused to the C terminus of MBP with linker 1 (MBP-linker1-MPR-TM) but could be obtained after changing the linker (MBP-linker2-MPR-TM). The crystal belongs to space group P32 with unit cell constants of a=172 Å, b=172 Å, c= 70 Å and alpha=beta=90 and gamma=120. The 2.5 Å crystal structure reveals the conformation of MBP and part of the linker region of this chimera, but the MPR-TM segment is unstructured.

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Acta Cryst. (2014). A70, C122
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The transmembrane subunit (gp41) of the envelope glycoprotein (Env) of HIV-1 associates non-covalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 is involved in many essential biological functions and its primary role is to anchor the Env in both viral and cellular membranes. Despite having many important biological functions, the atomic structure of gp41 TM domain remains unknown. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the prefusion or post-fusion conformations, which could not be recognized by the broadly neutralizing antibodies 2F5 and 4E10. Here we describe the expression, purification, biophysical characterization and crystallization of a chimera construct including maltose binding protein (MBP) and MPR-TM of gp41. The purified MBP-MPR-TM protein reacts with the broadly neutralizing antibodies 2F5 and 4E10 with nanomolar affinities and thereby may represent an immunologically relevant conformation mimicking a pre-hairpin intermediate of gp41. Crystals could not be obtained initially when MPR-TM was fused to the C terminus of MBP with linker 1 (MBP-linker1-MPR-TM) but could be obtained after changing the linker (MBP-linker2-MPR-TM). The crystal belongs to space group P32 with unit cell constants of a=172 Å, b=172 Å, c= 70 Å and alpha=beta=90 and gamma=120. The 2.5 Å crystal structure reveals the conformation of MBP and part of the linker region of this chimera, but the MPR-TM segment is unstructured.

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Acta Cryst. (2014). A70, C1141
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Membrane proteins are extremely difficult to crystallize, however they are highly important proteins for cellular function. Photosystem I, one of the most complex membrane proteins solved to date took more than a decade to have a structure solved to molecular resolution. Large, well-ordered crystal growth is one of the major bottlenecks in structural determination by x-ray crystallography, due to the difficulty of making the "perfect" crystal. The development of femtosecond nanocrystallography, which uses a stream of fully hydrated nanocrystals to collect diffraction snapshots, effectively reduces this bottleneck[1] Photosystem II changed our biosphere via splitting water and evolving oxygen 2.5 billion years ago. Using femtosecond nanocrystallography we are developing a time-resolved femtosecond crystallography method [2] to unravel the mechanism of water splitting by determining the conformational changes that take place during the oxygen evolution process. Multiple crystallization techniques were originally developed in order to make the nanocrystals necessary for femtosecond nanocrystallography. For Photosystem II nano/microcrystals a free interface diffusion method, is used to increase yield over traditional methods. These crystals are then characterized by three different methods before being used for collecting diff raction data. The three methods currently used are optical microscopy, dynamic light scattering (DLS), and Second Order Nonlinear Imaging of Chiral Crystals (SONICC).

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Acta Cryst. (2014). A70, C1147
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A prerequisite for conventional X-ray protein structure analysis is the growth of crystals with a sufficient size in the range of several µm. This is a time consuming and not always successful process, challenging especially when working with membrane proteins. The recently developed technique of femtosecond X-ray crystallography enables structure analysis of crystals with a size in the nm range, thus the process of growing large single crystals can be avoided. Moreover femtosecond X-ray nanocrystallography is a potential method to overcome the radiation damage problem and to perform time-resolved structure analysis (1, 2). Nanocrystals are too small to be detected with an optical microscope, hence crystal growth cannot be monitored with common methods used in crystallography. A powerful technique to screen for nanocrystals is Second Order Nonlinear Imaging of Chiral Crystals (SONICC). This method is based on the principle of second harmonic generation and detects noncentrosymmetric ordered crystals (3). However, the instrumentation is not generally accessible yet. In addition, proteins ordered in higher symmetry crystal classes do not necessarily lead to a positive SONICC signal. In this work, a new method is developed to screen for nanocrystals based on the reversibility of crystallization. We show that dilution experiments performed with a crystallization robot and monitored by a crystallization imaging system enable the distinction between precipitation comprised of nanocrystals and precipitation caused by aggregated protein.
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