|SDSU STRUCTURAL BIOLOGY PROGRAM|
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This week was the last meeting of the SDSU Structural Biology Program for the Fall Semester. Dr. Jeff Lee of The Scripps Research Institute shared his experiences in determining the x-ray crystal structure of the Ebola virus glycoprotein in complex with a monoclonal antibody derived from a survivor. During the course of his investigations, Dr. Lee developed methods for expression and purification of forty different glycoprotein constructs. Because of limiting amounts of material he relied on microfluidics to direct initial crystallization conditions and he improved diffraction quality by introducing the Fab fragment of a monoclonal antibody. To phase the data he first solved the structure of the free monoclonal Fab and combined this with Se-MAD data and density modification to generate surprisingly good experimental maps. The structure is beautiful as it shows not only where the functional amino acid sequences are located, but suggests possible mechanisms for proteolytic activation of the viral surface protein and structural rearrangement that could be responsible for fusion of the viral membrane with that of the host cell. It was a fitting end to a great semester of structural biology. Click here to see photos of this week's meeting.
This week we were privileged to host Dr. Case McNamara, a postdoctoral fellow at The Scripps Research Institute. Dr. McNamara shared the saga of his graduate research leading up to and including the determination of a difficult x-ray crystal structure of an active fragment from the Group A Streptococcus M1 coat protein. During the course of this research, Case overcame many practical complications including optimal design of protein constructs, preparation of diffraction quality crystals, phase determination by Se-MAD, and model building ambiguities. His efforts were rewarded once he was able to use his structural information to discover how this fascinating pathogenic protein has been optimized by nature to assume a dynamic coiled-coil structure. It was an excellent illustration of how creativity and persistence can overcome the hurdles one encounters on the path to structure determination by x-ray crystallography. Photos of this week's meeting are available here.
In this week's meeting of the San Diego State University Structural Biology Program we addressed the final hurdle that must be cleared for successful determination of a macromolecular structure by x-ray crystallography. That is, what do you do with a detailed molecular model once you final obtain it? Ultimately, the answer is that you use it to suggest testable hypotheses regarding how the molecule might function in biology. Dr. Huxford referred to this as "Structure-based biochemistry--the true killer app of x-ray crystallography". Check out this week's lecture slides here.
We will not be meeting next Wednesday November 26. Enjoy your Thanksgiving holiday! Our next meeting will be on Wednesday December 3 when we will have receive our enjoy listening to our first guest speaker share "true stories of x-ray structure determination". Be sure to drop by for some food and stay to listen to some great stories of how researchers have found creative ways to solve the problems encountered on their road to x-ray structure determination.Assignment
This week's meeting topic was Crystallographic Refinement. Dr. Huxford lead a discussion of this iterative process in which a molecular model is first built into electron density maps derived from experimental phases. Improved phase values can then be calculated and improved map density generated. Progress during this bootstrapping method can be monitored by measuring the agreement between the intensities observed during an experiment and structure factors calculated from the model atomic coordinates. We ended the meeting early this week and walked to Dr. Huxford's office where we watched model building and refinement carried out in real time. Check out the presentation slides and this week's homework assignment here.Assignment
This week we celebrated the promise of a new presidential administration by discussing the Phase Problem in macromolecular x-ray crystallography and how to go about solving it. Dr. Huxford introduced the math behind generating an electron density map and the fact that we do not directly measure a value of phase for each reflection spot during a diffraction experiment. He then introduced theoretical and practical aspects of the three principal experimental approaches commonly employed to calculate an initial estimate of phases and generate experimental electron density maps. Ask someone who attended this week's meeting about a "Difference Pattersons". Or even better, download Dr. Huxford's slides here.Assignment
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