We describe the recombinant expression and purification of VHHs and protein antigens, purification and analysis of protein complexes, crystallization, and optimization, X-ray structure determination by molecular replacement, and analysis of the complex. Here, we provide an updated procedure for determining and analyzing the X-ray structure of a VHH in complex with a protein antigen.
While multiple approaches exist for dissecting the interaction between a protein antigen and a VHH, X-ray crystallography remains the highest resolution method available. The majority of VHHs bind to folded proteins and thus recognize discontinuous three-dimensional epitopes. A detailed molecular analysis of VHH interactions with their cognate protein antigens is valuable for understanding structure-function relationships and for protein engineering. The evolutionary adaptations of VHHs have resulted in biophysical properties and antigen-binding modalities which are distinct from those of conventional antibodies. These proteins have generated considerable interest in a variety of applications as research reagents, crystallization chaperones, and therapeutics. VHHs are antigen-binding domains cloned from heavy-chain antibodies found in camelids. It is our hope that these validation resources will help the cryo-EM community to obtain a better understanding of the quality and of the best ways to assess the quality of cryo-EM structures in EMDB and PDB. The output of all three tiers is publicly available, either through the EMDB website (tiers 1 and 2) or through the wwPDB ftp sites (tier 3), although the content of all three will evolve over time (fastest for tier 1 and slowest for tier 3). The details of the new resource are described, with an emphasis on the tier 1 system. Successful metrics from tier 2 will be proposed for inclusion in the wwPDB validation pipeline and reports. Tier 3, finally, consists of the validation reports and servers that are produced by the Worldwide Protein Data Bank (wwPDB) Consortium. As more experience is gained with the metrics included in the VA resource, it is expected that consensus will emerge in the community regarding a subset that is suitable for inclusion in the tier 2 system.
#VINYLSTUDIO ERROR 32 THR ARCHIVE#
These metrics are presented on the entry-specific web pages for the entire archive on the EMDB website. Tier 2 comprises a subset of the validation metrics covered by the VA resource that have been subjected to extensive testing and are considered to be useful for specialists as well as nonspecialists. This system is particularly useful for cryo-EM experts, both to validate individual structures and to assess the utility of new validation metrics. Tier 1 covers an extensive and evolving set of validation metrics, including tried and tested metrics as well as more experimental ones, which are calculated for all EMDB entries and presented in the Validation Analysis (VA) web resource. Based on recommendations from community experts, the validation resources are developed in a three-tiered system. With input from the community, EMDB has developed new resources for the validation of cryo-EM structures, focusing on the quality of the volume data alone and that of the fit of any models, themselves archived in the Protein Data Bank (PDB), to the volume data. The Electron Microscopy Data Bank (EMDB) is the central archive of the electron cryo-microscopy (cryo-EM) community for storing and disseminating volume maps and tomograms.
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