Automated protein crystal structure determination using ELVES

Abstract

Efficient determination of protein crystal structures requires automated x-ray data analysis. Here, we describe the expert system ELVES and its use to determine automatically the structure of a 12-kDa protein. Multiwavelength anomalous diffraction analysis of a selenomethionyl derivative was used to image the Asn-16-Ala variant of the GCN4 leucine zipper. In contrast to the parallel, dimeric coiled coil formed by the WT sequence, the mutant unexpectedly formed an antiparallel trimer. This structural switch reveals how avoidance of core cavities at a single site can select the native fold of a protein. All structure calculations, including indexing, data processing, locating heavy atoms, phasing by multiwavelength anomalous diffraction, model building, and refinement, were completed without human intervention. The results demonstrate the feasibility of automated methods for determining high-resolution, x-ray crystal structures of proteins.

Fig. 1.

Levels of automation in elves. elves automates widely used crystallographic software (Lower) that are currently run by means of user-specified scripts. Green wedges indicate the scope of each elves program. Four programs make up the first level of elves automation. wedger processes a single wedge of data by using mosflm (6). scaler performs local scaling (7) and merges multiple wedges of data by using the indicated programs. phaser locates heavy atoms with shelx (8) or rantan (5) and refines and searches for additional heavy atoms with mlphare (9). mlphare and dm (10) are used to calculate phases, and the electron density map is calculated with the CCP4 suite (5). Scripts for o (11), arp/warp (4), refmac (12), and cns (13) are produced. refmacer uses arp/warp and refmac to build and refine a molecular model. These programs are coordinated by the next level of automation, called processer, which also runs solve (2) and arp/warp (4). The elves main program runs processer in the fully automated mode.

Fig. 2.

Experimental, 1.8-Å resolution MAD-phased electron density map (P3₁21, contoured at 1 σ) produced by elves superimposed on the refined model of the GCN4 Asn-16-Ala leucine-zipper variant. (A) Cross section through the trimer at the level of the Ala-16-Leu-12-Leu-12 layer. (B) Cross section through the trimer at the level of the Leu-12-Ala-16-Ala-16 layer.

Fig. 3.

The Asn-16-Ala variant of the GCN4 leucine zipper forms an antiparallel, trimeric coiled coil. (A) Stereo ribbon diagram of the overall structure. Each helix is colored in increasingly cool colors from the amino to the carboxyl terminus. (B) Superpositions of the structures determined by automated (blue) and manual (yellow) methods in the trigonal (Left) and tetragonal (Right) crystal forms. (C) Cross section showing the Ala-16-Leu-12-Leu-12 layer with the van der Waals surfaces of the core amino acids filling the space in the core of the trimer. (D) Close packing of the Leu-12-Ala-16-Ala-16 layer. (E) In contrast, a 165-ų cavity exists in the Ala-16 layer of the parallel trimer stabilized by benzene (29).