infectious. The crowded environment in the 100-nm sized viral particle makes it impossible for the AVP proteins to utilise regular three-dimensional diffusion to find the large number of protease targets. Instead, adenovirus proteins em- ploy a small peptide pVIc as a ‘‘molecular sled’’, which allows for one- dimensional sliding along DNA. We use single-molecule techniques to verify the one-dimensional movement model of pVIc peptide and to decipher the mo- lecular mechanisms underlying adenovirus maturation. 917-Pos Board B686 Single Molecule Optical Determination of Bestrophin Stoichiometry Shashank Bharill, Zhu Fu, Ehud Y. Isacoff. University of California Berkeley, Berkeley, CA, USA. Best macular dystrophy (BMD) is an autosomal dominant form of macular de- generation, linked to mutations in the hBEST1 gene, which encodes the cal- cium activated chloride channel (CACC) bestrophin-1. The bestrophin family includes three additional members: hBEST2, 3 and 4. Because mutations in hBEST1 cause BMD, but a knock-out does not, hBEST1 mutants have been suggested to exert a dominant negative effect through interaction with other CACCs. Using single molecule subunit counting and co-localization we find that each hBEST forms a homotetrameric channel. Despite considerable con- servation among hBESTs, hBEST1 has little or no interaction with other hBESTs. Deletion and chimera analysis are being used to identify the portions of hBEST1 that allow assembly of like subunits and prevent assembly with other hBESTs. Our results suggest that the pathology caused by hBEST1 mu- tations is not due to assembly with other CACCs. 918-Pos Board B687 Incremental Phosphorylation of a Dynamic Lawn of NDC80 Complexes Provides Graded Control of Kinetochore-Microtubule Affinity Anatoly Zaytsev 1,2 , Lynsie J.R. Sundin 3 , Boris Nikashin 1 , Keith F. DeLuca 4 , Jeanne Mick 3 , Geoffrey J. Guimaraes 3 , Fazly I. Ataullakhanov 1 , Ekaterina L. Grishchuk 2 , Jennifer G. DeLuca 3 . 1 CTPPhChPh RAS, Moscow, Russian Federation, 2 University of Pennsylvania, Philadelphia, PA, USA, 3 Colorado State University, Fort Collins, CO, USA, 4 Colorado State University, Fort Collins, UT, USA. The stability of kinetochore-microtubule (KMT) attachments is finely tuned to drive different mitotic processes. This regulation involves phosphorylation of NDC80 complex, a major component of the MT-binding interface at kineto- chores. The fundamental question of how the number and stability of KMT at- tachments is regulated by NDC80 phosphorylation remains unanswered. The Hec1 subunit of the NDC80 complex has an unstructured ‘‘tail,’’ which is re- quired for KMT attachment in vivo and contributes to the NDC80 complex- MT binding in vitro. This tail is an established target for Aurora kinases and has 9 mapped phosphorylation sites. To understand how phosphorylation of the Hec1 tail affects MT-binding characteristics of single NDC80 complexes we expressed and purified NDC80Bonsai complexes with different number of phospho-mimetic mutations in Hec1 tail. With TIRF microscopy we found that Hec1 tail phosphorylation leads to a graded increase in NDC80 diffusion and the shortening of its MT residency time, but cooperativity of NDC80- MT binding is only weakly affected by Hec1 tail phosphorylation. To under- stand physiological relevance of the phosphoregulation of NDC80 complexes we used computational approaches to model kinetochore-MT interface contain- ing multiple NDC80 molecules. We show that the behavior of such interface strongly depends on the spatial organization of the NDC80 complexes. KMT interface that contained ‘‘repetitive sites’’ of NDC80 complexes greatly ampli- fied the relatively small, phosphorylation-induced changes in the residency time of single NDC80. However, the KMT interface that contained a dynamic ‘‘lawn’’ of un-clustered and uncoordinated NDC80 complexes exhibited a graded response to phosphorylation and produced an excellent fit to our data with cells in prometaphase and metaphase. We conclude that incremental phosphorylation of NDC80 complexes drives the graded regulation of kinetochore-microtubule affinity during mitotic progression. Biomolecular NMR Spectroscopy 919-Pos Board B688 Solution Structure and Dynamics of a Plant Pathogen Effector James L. Tolchard 1 , Vicki S. Chambers 1 , Laurence S. Boutemy 2 , Mark J. Banfield 2 , Tharin M.A. Blumenschein 1 . 1 University of East Anglia, Norwich, United Kingdom, 2 John Innes Centre, Norwich, United Kingdom. Plant diseases account for at least $220 billion of crop losses worldwide every year, and have a significant impact on global food security and bio-fuel avail- ability. Bacterial, oomycetes and fungi pathogens secrete protein effectors into plant cells, where they perturb cellular processes, presumably to the benefit of the pathogen. In oomycetes, an N-terminal protein motif (RXLR) is important for targeting plant pathogen effectors into host cells (Whisson et al. 2007, Na- ture450, 115 and Oliva et al. 2010, Cell. Microbiol.12, 1015). However, little is known about the molecular mechanisms by which RXLR effectors manipu- late host cell pathways. We have been studying the RXLR effector AVR3a11 (from Phytophthora capsici, a pathogen of pepper), which shares sequence similarity with the well-studied AVR3a effector from the Irish potato famine pathogen Phytophthora infestans (the causative agent of late blight in potato and tomato). Using a combination of 2D and 3D nuclear magnetic resonance (NMR) spectroscopy experiments, 75% of the AVR3a11 backbone has been assigned. With additional 13 C-HSQC-NOESY and 15 N-HSQC-NOESY exper- iments, a structural ensemble model has been generated which is in good agreement with the X-ray structure of AVR3a11 (PDB code 3ZR8, Boutemy et al. 2011, J. Biol. Chem.286, 35834). Backbone amide T 1 ,T 2 and heteronu- clear NOE relaxation experiments at 800 MHz, show that AVR3a11 behaves as expected for a well ordered protein. However, the measured transverse re- laxation is faster than theoretically expected for a protein this size, suggesting that conformational exchange may be occurring. Additional information on conformational exchange was obtained from hydrogen/deuterium exchange experiments. 920-Pos Board B689 E. Coli F1Fo ATP Synthase Subunit C: Solution NMR Structure by CS-Rosetta Yisong Tao, Mark Girvin. Albert Einstein College of Medicine, Bronx, NY, USA. The ATP synthase is the main source of ATP in cells, whose function is to inter-convert chemical energy and transmembrane electrochemical potential difference. The enzyme contains two domains: a soluble F 1 domain where ATP is synthesized/hydrolyzed and a transmembrane F O domain, which func- tions as an ion pump. Multiple (9-15) copies of subunit c form an oligomeric ring in the F O domain. The ion translocation activity takes place at the inter- face of c-ring and subunit a where an essential acidic residue in middle of the outer helix in subunit c undergoes a protonation/deprotonation process, which couples with the relative rotation of c-ring to subunit a. Although high- resolution X-ray structures of the c-rings from several organisms have been solved, the mechanism of ion translocation still needs to be clarified at atomic resolution. We aim to solve the structures of E. coli subunit c at both protonation states of the essential Asp61 and elucidate any conformational changes at the active site during the ion translocation process. Due to the difficulty of obtaining unam- biguous long-range NOE constraints in membrane proteins, traditional solution NMR methods were unsuccessful in this case. We employed CS-Rosetta, which utilizes minimal NMR constraints, in our study. Our study of the subunit c in the protonated state shows that the choice of proper scoring weights is essential in the application of CS-Rosetta to mem- brane proteins. Using limited NMR restraints this method converges on a struc- ture for E. coli subunit c that is similar to the X-ray structures of subunit c from other organisms. Further improving the quality of the structure may be achieved by optimizing the weights in the calculation. We are also using this approach on deprotonated subunit c and hoping to model the c-ring from our results. 921-Pos Board B690 NMR Solution Structure of Opa60: A Neisserial Membrane Protein that Mediates Host Phagocytosis Daniel A. Fox, Linda Columbus. University of Virginia, Charlottesville, VA, USA. The family of Opa proteins from Neisseria gonorrhoeae and N. meningitidis are eight-stranded b-barrel outer membrane proteins that induce human cells to en- gulf the bacterium by engaging three different host receptors: carcinoem- bryonic antigen cellular adhesion molecules (CEACAM), heparansulfate proteoglycans (HSPG), or integrins via HSPG and fibronectin or vitronectin. The receptor engaged depends on the sequence of two of the extracellular loops, hypervariable loops 1 and 2, which are highly variable between isolates. Multiple sequence alignment of the HV loops does not reveal specificity motifs among the family of Opa proteins due to the extreme variability in the amino acid sequences. To investigate the determinants of Opa-receptor interactions, the NMR solution structure of Opa60, which engages CEACAM receptors, was determined. In order to solve the structure, a suite of three dimension NMR experiments were performed to obtain an assignment, each optimized for the different Sunday, February 3, 2012 179a