The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.T he emergence of X-ray free electron laser (X-FEL)-based serial femtosecond crystallography holds the promise of solving the 3D structure of proteins that can only crystallize as "nanocrystals" (NCs) or are highly sensitive to radiation damage (1-5). NCs appropriate for X-FEL experiments are considered to be 200 nm to 2 μm in size (6). This size is constrained primarily by the requirements of the NC delivery system to the X-FEL beam. In addition to allowing for structure resolution of NCs by X-FEL experiments, they provide the advantage of requiring no crystal cryoprotection because these experiments are performed at room temperature (3, 7). Given the opportunities that X-FELs offer to the field of crystallography, efficient methodologies to detect NCs from single crystallography drops and to optimize these identified conditions yielding NCs will be essential for future developments in structural biology. Current methods to detect the presence of NCs include dynamic light scattering (DLS), bright-field microscopy, birefringence microscopy, and intrinsic tryptophan UV fluorescence imaging, as well as technologies that rely upon second harmonic generation, such as second order nonlinear imaging of chiral crystals (SONICC) (8, 9) and X-ray powder diffraction. However, limitations of these imaging techniques include (i) ineffective detection of crystals smaller than 5 μm (8, 10), (ii) false-positive conditions as a result of interference from precipitate backgrounds (8, 10), and (iii) false-negative conditions resulting from the lack of tryptophan residues in the case of UV fluorescence and from the lack of chiral centers in the case of SONICC (11). Although DLS can accurately measure the size distribution of nanometer-sized protein aggregates, it is unable to distinguish unambiguously between amorphous and crystalline (12). Finally, X-ray powder diffraction, a method that has been applied to evaluate samples for the presence and concentration of NCs, requires more material than is produced in a single crystallization screening d...

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Antibody-dependent cellular cytotoxicity (ADCC) is a host defense mechanism in which Fc receptor-bearing effector cells in combination with antigen-specific antibodies recognize and kill antigen-expressing target cells. The authors previously described a murine monoclonal antibody (MAb-ID6) that mediated ADCC activity against HIV-infected cells. It was demonstrated that the specificity of MAb-ID6 maps to the first 204 amino acids of gp120; however, the exact epitope was not identified. In the present work, by screening phage display libraries with MAb-ID6, the authors have mapped the corresponding epitope to amino acids 86-100 (HIV-1 gp120 sequence). This epitope lies within the C1 region of gp120 and is highly conserved among all subtypes and circulating recombinant forms of HIV-1. Thus, these phage mimotopes of C1 may serve as components of a vaccine for the induction of gp120-specific antibodies mimicking MAb-ID6.