Dr. Kenneth A. Taylor

Our research uses 3-D electron microscopy (EM) to study structure and function at the resolution of molecules and supramolecular assemblies both isolated and within cells. This aspect of EM includes techniques such as electron crystallography, electron tomography, helical and &ldquo;single particle&rdquo; 3-D image reconstruction. Since we specialize in EM, we also spend considerable effort in method development particularly as it pertains to electron tomography (ET). Present efforts at method development are aimed at algorithms to improve the molecular images that we can obtain by ET.<span> <span style="mso-spacerun: yes;">&nbsp;</span></span>We moved from an emphasis on electron crystallography of 2-D arrays to ET to improve our 3-D images obtained from fast frozen, active insect flight muscle (IFM). Rapid freezing was obtained by smashing the tissue against a liquid helium cooled copper mirror. The tissue was then freeze substituted for subsequent sectioning and 3-D imaging. To study actively contracting IFM, we needed images of individual cross-bridges and that is something that cannot be obtained by any of the other 3D-EM techniques. To improve signal-to-noise ratio in these 3-D images but still maintaining the ability to distinguish different structures within the muscle lattice, we adapted methods for classifying images that were developed for single particle 3-D reconstruction to identify and group self-similar 3-D structures for subsequent averaging. These methods enabled us for the first time to study the conformations of tension generating cross-bridges in situ. <p><span><span style="font-size: 9.0pt; font-family: 'Arial',sans-serif;">The culmination of this work is its application to time resolved studies of contracting muscle.<span> This effort applies the combination of 3-D tomography, X-ray diffraction, muscle mechanics and fast freezing to trap force-bearing cross-bridges in situ.<span> These experiments allow us to build atomic models of the different cross-bridge forms using the crystal structures of myosin and filament structures of actin.<span> Our first effort shows cross-bridges at all stages of the working stroke as well as cross-bridges in the presumably weak binding states that precede force production.<span> The work suggests that the position of the myosin motor domain on actin changes during the so-called weak to strong binding transition in addition to the well-known movements of the light chain domain lever arm during the powerstroke itself. </span></span></span></span></span></span></p> <p><span><span style="font-size: 9.0pt; font-family: 'Arial',sans-serif;">Currently, our research emphasis is the structure of the thick, myosin-containing filaments from striated muscle. In 2016 we published the first 3D image of such a filament at subnanometer resolution that revealed for the first time the arrangement of the myosin coiled-coil tails within the filament backbone. We have obtained subnanometer resolution reconstructions of two other species of insect that use this type of muscle. In 2021 our resolution reached 4.2&Aring;, sufficient to build the first atomic model of a myosin tail within its native environment. That atomic model is the longest 2-stranded coiled coil in the Protein Data Bank. Current thick filament work has reached 2.7&Aring; resolution producing an even better atomic model. <span style="mso-spacerun: yes;">&nbsp;</span></span></span></p> <p><span style="font-size: 9.0pt; font-family: 'Arial',sans-serif;">Although we will continue to work on atomic resolution reconstructions of muscle filaments, our plan is to develop a second line of investigation utilizing the new technique of Focused Ion Beam (FIB) milling. FIB milling produces frozen-hydrated tissue specimens that are thin enough for high resolution imaging, without the use of chemical fixation, dehydration, plastic embedding and sectioning, all processes that can deform the tissue and create artifacts. When applied to muscle tissue, it will facilitate 3-D imaging of all parts of the muscle without pretreatment. We can then repeat the work on active muscle contraction mentioned above but this time without the freeze substitution and sectioning. This will produce more detailed images of active muscle contraction and facilitate studies of other parts of the muscle such as the Z-disk, sarcolemma, T-tubules among others. We obtained one results using this technique, which required the interested graduate student to spend a year at Yale University in a former postdoc&rsquo;s laboratory which was equipped with a FIB milling machine. To move the FIB effort along, the PI will take a sabbatical at the Max Planck Institute of Biochemistry in Martinsried, Germany for the academic year 2024-2025. The MPIB is the major site of development of the FIB milling technique for biological specimens.</span></p>

31. Abbasi Yeganeh F, Rastegarpouyani H, Li J, Taylor KA. Structure of the Drosophila melanogaster Flight Muscle Myosin Filament at 4.7 &Aring; Resolution Reveals New Details of Non-Myosin Proteins. <i>Int J Mol Sci</i>. 2023;24(19):14936. PubMed PMID: doi:10.3390/ijms241914936. 32. Hojjatian A, Taylor DW, Daneshparvar N, Fagnant PM, Trybus KM, Taylor KA. Double-headed binding of myosin II to F-actin shows the effect of strain on head structure. <i>J Struct Biol</i>. 2023;215(3):107995. Epub 20230704. doi: 10.1016/j.jsb.2023.107995. PubMed PMID: 37414375; PMCID: PMC10544818. 33. Li J, Rahmani H, Abbasi Yeganeh F, Rastegarpouyani H, Taylor DW, Wood NB, Previs MJ, Iwamoto H, Taylor KA. Structure of the Flight Muscle Thick Filament from the Bumble Bee, <i>Bombus ignitus</i>, at 6 A Resolution