National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
5T32GM008496
米国
Department of Energy (DOE, United States)
DE-FC02-02ER63421
米国
National Science Foundation (NSF, United States)
DMR-1548924
米国
Sao Paulo Research Foundation (FAPESP)
16/24191-8
ブラジル
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35 GM128867
米国
Spanish Ministry of Economy and Competitiveness
BIO2015-64216-P
スペイン
Sao Paulo Research Foundation (FAPESP)
17/13485-3
ブラジル
Spanish Ministry of Economy and Competitiveness
MDM2014-0435-01
スペイン
Spanish Ministry of Economy and Competitiveness
PGC2018-101370-B-100
スペイン
引用
ジャーナル: Acta Crystallogr D Struct Biol / 年: 2020 タイトル: Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER. 著者: Logan S Richards / Claudia Millán / Jennifer Miao / Michael W Martynowycz / Michael R Sawaya / Tamir Gonen / Rafael J Borges / Isabel Usón / Jose A Rodriguez / 要旨: Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective ...Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose.