The Imaging Sciences Laboratory is involved in a major collaborative research effort with the Institutes involving the use of image processing techniques and advanced computational techniques in structural biology to analyze electron micrographs and NMR spectra with the goal of determining macromolecular structures and dynamics. Recent efforts have concentrated on the 3D reconstruction, analysis and interpretation of the structures of icosahedral virus capsids in addition to structure determination and analysis of isolated and complexed proteins and nucleic acids. Ongoing research involves analyses of structures related to herpesvirus and papillomairus and other icosahedral virus capsids. Papillomaviruses (e.g. HPV-16) encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein L2 is not required for capsid formation, it is thought to participate in encapsidation of the viral genome, and plays a number of essential roles in the viral infectious entry pathway. Cryo-electron microscopy and difference 3D reconstruction analysis of purified capsids revealed an icosahedrally-ordered L2-specific density beneath the axial lumen of each L1 capsomer. We have used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV-16 capsids assembled in 293T cells. The major capsid protein, L1, initially forms a loosely connected procapsid which, under in vitro conditions, condenses over several hours into the more familiar 60 nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by 5% in diameter;its pentameric capsomers change in structure, most markedly in their axial region;and the interaction surfaces between adjacent capsomers are consolidated. These structural changes are accompanied by the formation of disulfide crosslinks that enhance the stability of the mature capsid. At slightly basic pH, the capsids do not achieve compete disulfide bond formation (e.g. maturation). Simply buffering the lysate to more neutral conditions allowed the production of recombinant capsids with >90% disulfide bonds. Cryo-EM with image reconstruction revealed that more fully mature recombinant HPV16 capsids exhibited a much greater degree of regularity compared to HPV16 capsids produced using standard procedures. Their greater regularity allowed us to reconstruct the capsid to higher resolution. sufficient to allow robust fitting of the L1 crystal structure into the density map. The ability to produce fully mature recombinant capsids should benefit further structural investigations of native HPV capsids. Moreover, fully mature pseudovirions should be viewed as preferred reagents for studies aimed at elucidating the entry pathways used by HPV in the course of natural infections. The C175S mutant, which does not crosslink, shows similar maturation-related structural changes but capsids are significantly larger, under otherwise similar conditions. We conclude that the observed structural size changes facilitates maturation, but crosslink formation is required to lock the capsid into the mature state. These results will submitted for publication and are partially discussed in a new book chapter (Buck CB, Trus BL (2012) The papillomavirus virion: a machine built to hide molecular Achilles heels. In Adv Exp Med Biol 726:403-22). We have been collaborating in a long-term study of RNA containing viruses. In one study published last year (J Virol 86:6470-80) the T=3 capsid structure of Rabbit Hemorrhagic Disease Virus (RHDV) was studied to 8A resolution. Using X-ray structures from similar viruses, we were able to fit the (high-resolution) X-ray structure in the EM density creating a pseudo atomic model. Mutagenesis experiments were used to demonstrate that changes in the N terminus could change the packing, size, and symmetry of the VP1 capsid protein to produce T = 4 capsids. In another RNA containing virus study published this year (J Virol 86:8314-8) cryoelectron microscopy reconstruction of Cryphonectria nitschkei virus 1, a double-stranded RNA (dsRNA) virus, shows that the capsid protein (60 copies/particle) is formed by a repeated helical core, indicative of gene duplication. This structure is similar to totivirus L-A capsids suggesting a shared motif for 120-subunit T=1 capsids. We have also been developing computational tools for the study of the structure and dynamics of biological macromolecules using NMR data. We develop and maintain the Xplor-NIH software package for structure determination, which is used in the NMR labs in the Institutes, and also worldwide. Development of Xplor-NIH has continued in the following areas: (a) improved methods for treating SAXS and NMR RDC data in studying mixtures of small oligomers of theHIV capsid protein;(b) development of methods in solid state NMR applied to the study of amyloid beta structures;(c) much improved use of statistical potentials for general use in protein and nucleic acid structure determination.

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Luque, Daniel; Gómez-Blanco, Josué; Garriga, Damiá et al. (2014) Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage. Proc Natl Acad Sci U S A 111:7641-6
Tian, Ye; Schwieters, Charles D; Opella, Stanley J et al. (2014) A practical implicit solvent potential for NMR structure calculation. J Magn Reson 243:54-64
Schwieters, Charles D; Clore, G Marius (2014) Using small angle solution scattering data in Xplor-NIH structure calculations. Prog Nucl Magn Reson Spectrosc 80:1-11
Cardone, Giovanni; Moyer, Adam L; Cheng, Naiqian et al. (2014) Maturation of the human papillomavirus 16 capsid. MBio 5:e01104-14
Yu, Xiaozhen; Wu, Xiongwu; Bermejo, Guillermo A et al. (2013) Accurate high-throughput structure mapping and prediction with transition metal ion FRET. Structure 21:9-19
Jaremko, Mariusz; Jaremko, Lukasz; Kim, Hai-Young et al. (2013) Cold denaturation of a protein dimer monitored at atomic resolution. Nat Chem Biol 9:264-70
Yan, Si; Hou, Guangjin; Schwieters, Charles D et al. (2013) Three-dimensional structure of CAP-gly domain of mammalian dynactin determined by magic angle spinning NMR spectroscopy: conformational plasticity and interactions with end-binding protein EB1. J Mol Biol 425:4249-66
Buck, Christopher B; Day, Patricia M; Trus, Benes L (2013) The papillomavirus major capsid protein L1. Virology 445:169-74
Montelione, Gaetano T; Nilges, Michael; Bax, Ad et al. (2013) Recommendations of the wwPDB NMR Validation Task Force. Structure 21:1563-70
Ryabov, Yaroslav; Suh, Jeong-Yong; Grishaev, Alexander et al. (2009) Using the experimentally determined components of the overall rotational diffusion tensor to restrain molecular shape and size in NMR structure determination of globular proteins and protein-protein complexes. J Am Chem Soc 131:9522-31

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