SEQUENCE SLIDER: expanding polyalanine fragments for phasing with multiple side-chain hypotheses

Abstract

Fragment‐based molecular‐replacement methods can solve a macromolecular structure quasi‐ab initio. ARCIMBOLDO, using a common secondary‐structure or tertiary‐structure template or a library of folds, locates these with Phaser and reveals the rest of the structure by density modification and autotracing in SHELXE. The latter stage is challenging when dealing with diffraction data at lower resolution, low solvent content, high β‐sheet composition or situations in which the initial fragments represent a low fraction of the total scattering or where their accuracy is low. SEQUENCE SLIDER aims to overcome these complications by extending the initial polyalanine fragment with side chains in a multisolution framework. Its use is illustrated on test cases and previously unknown structures. The selection and order of fragments to be extended follows the decrease in log‐likelihood gain (LLG) calculated with Phaser upon the omission of each single fragment. When the starting substructure is derived from a remote homolog, sequence assignment to fragments is restricted by the original alignment. Otherwise, the secondary‐structure prediction is matched to that found in fragments and traces. Sequence hypotheses are trialled in a brute‐force approach through side‐chain building and refinement. Scoring the refined models through their LLG in Phaser may allow discrimination of the correct sequence or filter the best partial structures for further density modification and autotracing. The default limits for the number of models to pursue are hardware dependent. In its most economic implementation, suitable for a single laptop, the main‐chain trace is extended as polyserine rather than trialling models with different sequence assignments, which requires a grid or multicore machine. SEQUENCE SLIDER has been instrumental in solving two novel structures: that of MltC from 2.7 Å resolution data and that of a pneumococcal lipoprotein with 638 residues and 35% solvent content.

Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate N -Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development

“…The first Cp GNA1 structure, determined from crystals belonging to the space group P2 1 2 1 2, with one monomer in the asymmetric unit, was solved by using the multisolution parallel phasing software ARCIMBOLDO ( 25 ). Extension of the placed fragments with SEQUENCE SLIDER ( 26 ) was required. The dimeric structure of Cp GNA1 in complex with acetyl-CoA and glucose-6-phosphate (Glc6P) from data at 1.95 Å in space group P2 1 2 1 2 1 was solved by molecular replacement with Phaser ( 27 ) using the monomeric structure ( Fig.…”

“…The search was set to locate 1 copy of an ensemble generated with an alpha version of the software ALEPH ( 51 ) The ensemble of two models represented the probable fold composed by 5 beta strands flanked by 2 alpha helices at each side and it contained the GNA1 of Saccharomyces cerevisiae (PDB id 1I21 , 19% identity to target sequence) and the acetyltransferase from Agrobacterium tumefaciens (PDB 2DXQ , 26% identity to target sequence). The ARCIMBOLDO_LITE solution was completed by SEQUENCE SLIDER ( 26 ) using side chain modeling with Scwrl4 ( 52 ), refinement with REFMAC5 ( 53 ), and expansion with SHELXE, resulting in a best trace of 120 residues with CC 31.9. The structure of Cp GNA1 with the substrates at 1.95 Å, in space group P2 1 2 1 2 1 , containing two monomers in the asymmetric unit was phased by molecular replacement ( 27 ) using the previous model.…”

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources

“…The side chain evaluator method called SEQUENCE SLIDER (SLIDER) provides a novel framework to coordinate this complex task of integrating structural and genetic information. SLIDER ( 48 ) was first created in the ab initio phasing scope of ARCIMBOLDO methods ( 49 ), in which small fragments are identified in the asymmetric unit using molecular replacement ( 50 ) and the rest of the structure is revealed through density modification and automatic map interpretation ( 51 , 52 ). SLIDER uses available sequence information, secondary structure prediction or alignment between remote homologues, to build the most probable side-chain atoms into a partial solution usually composed of polyalanine fragments.…”

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural source

“…The side chain evaluator method called SEQUENCE SLIDER (SLIDER) provides a framework to model this complex task of integrating structural and genetic information. SLIDER (46) was first created in the ab initio phasing scope of ARCIMBOLDO method (47), in which small fragments are identified in the asymmetric unit using Molecular Replacement (48) and the rest of the structure is revealed through density modification and automatic map interpretation (49).…”

“…SLIDER uses available sequence information, secondary structure prediction or alignment between remote homologues, to build most probable side-chain atoms into partial solution usually composed of polyalanine fragments, a discrimination in global statistics of the agreement between data and model may reveal true sequence hypothesis (46). Herein, we describe SLIDER, implemented to integrate structure determination, mass spectrometry and genetic information, to address the heterogeneity of complex samples purified from natural sources building a probability of amino acids per residue basis.…”