Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER

Author(s)

Richards, Logan S. ; Millan, Claudia ; Miao, Jennifer ; Martynowycz, Michael W. ; Sawaya, Michael R. ; Gonen, Tamir ; Borges, Rafael J. ; Uson, Isabel ; Rodriguez, Jose A.

• Document type: Journal article
• Source: ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY; v. 76, p. 10-pg., 2020-08-01.

Abstract

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 ofARCIMBOLDO, a suite of programs which employs thisab initioalgorithm for phase determination. Here, the use ofARCIMBOLDOis investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of theARCIMBOLDO_SHREDDERpipeline to provide phasing solutions for a structure of proteinase K from 1.6 angstrom resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%.ARCIMBOLDO_SHREDDERidentified 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 inPhaserto 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 usingSHELXE. 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. (AU)

• FAPESP's process: 16/24191-8 - Development of methods for crystallographic structure elucidation and structural studies of toxic mechanism of snake venom Phospholipases A2 homologue proteins
• Grantee: Rafael Junqueira Borges
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 17/13485-3 - Development and release of SEQUENCE SLIDER: a multi-side chain evaluator applied to toxinology and phasing
• Grantee: Rafael Junqueira Borges
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor