Making a difference in multi-data-set crystallography: simple and deterministic data-scaling/selection methods

Abstract

<jats:p>Phasing by single-wavelength anomalous diffraction (SAD) from multiple crystallographic data sets can be particularly demanding because of the weak anomalous signal and possible non-isomorphism. The identification and exclusion of non-isomorphous data sets by suitable indicators is therefore indispensable. Here, simple and robust data-selection methods are described. A multi-dimensional scaling procedure is first used to identify data sets with large non-isomorphism relative to clusters of other data sets. Within each cluster that it identifies, further selection is based on the weighted ΔCC<jats:sub>1/2</jats:sub>, a quantity representing the influence of a set of reflections on the overall CC<jats:sub>1/2</jats:sub> of the merged data. The anomalous signal is further improved by optimizing the scaling protocol. The success of iterating the selection and scaling steps was verified by substructure determination and subsequent structure solution. Three serial synchrotron crystallography (SSX) SAD test cases with hundreds of partial data sets and one test case with 62 complete data sets were analyzed. Structure solution was dramatically simplified with this procedure, and enabled solution of the structures after a few selection/scaling iterations. To explore the limits, the procedure was tested with much fewer data than originally required and could still solve the structure in several cases. In addition, an SSX data challenge, minimizing the number of (simulated) data sets necessary to solve the structure, was significantly underbid.</jats:p>

The Crystal Structure of Bromide-BoundGtACR1 Reveals a Pre-Activated State in the Transmembrane Anion Tunnel

“…Data were processed with XDS and scaled and merged with XSCALE (Kabsch, 2010a;Kabsch, 2010b) . The data sets were further selected to improve the final merging data set with the XDSCC12 (Assmann et al, 2020). 36 partial data sets were collected, processed, and merged to a final data set to 3.2 Å resolution, in which 31 data sets were collected on the crystals in the IMISX setup and 5 data sets were collected from crystals harvested on loop.…”

De novo crystal structure determination of double stranded RNA binding domain using only the sulfur anomalous diffraction in SAD phasing

“…Favorable factors included a large (15%) sulfur content (i. e. percentage of sulfur-containing residues in the polypeptide chain), the presence of disulfide bonds, which account as “super-sulfur” sites ( Kraatz et al., 2018 ), and diffraction to high resolution (1.5 ​Å). Later developments in diffraction hardware and crystallographic programs, together with extended and judicious use of data statistics ( Foos et al., 2019 ; Assmann et al., 2020 ) made native-SAD phasing accessible to numerous other proteins ( Liu et al., 2012 ; Liu and Hendrickson, 2015 ; Rose et al., 2015 ). The absorption edge of sulfur is λ ​= ​5.02 ​Å.…”

“…Among the various synchrotron data-collection strategies that have been proposed to optimize native-SAD ( Olieric et al., 2016 ) - including sulfur-SAD - phasing, the use of a long-wavelength (2.7 ​Å) over 1.9 ​Å ( Cianci et al., 2016 ; Banerjee et al., 2016 ; Basu et al., 2019b ) was shown to be advantageous for 100 ​μm or smaller crystals ( Basu et al., 2019b ; Liebschner et al., 2016 ). Moreover, successful native SAD phasing was achieved in several difficult cases using low X-ray dose, high redundancy and multi-orientations data collection of multiple crystals ( Liu et al., 2012 , 2014 ; Assmann et al., 2020 ; Banerjee et al., 2016 ; Akey et al., 2014 ; El Omari et al., 2014 ; Klinke et al., 2015 ), or even a single crystal ( Weinert et al., 2015 ; Basu et al., 2019 , Basu et al., 2019 ), which obviously removes the problem of non-isomorphism between various crystals. The latest technical advances include solution-free mounting systems to minimize X-ray absorption at long-wavelengths ( Yu et al., 2020 ) and assembly of numerous partial single-crystal data sets from microcrystals with sizes less than 10 ​μm ( Cianci et al., 2019 ; Guo et al., 2019 ; Nass et al., 2020 ).…”

An automated platform for structural analysis of membrane proteins through serial crystallography

“…-CC 1/2 iteratively (Assmann et al, 2020). For in situ datasets collected at PetraIII P14 beamline, we used an in-house script to run CrystFEL (White et al, 2012) version 0.8.0 automatically as the data had arrived.…”

“…ACER3-Cryo: 425 sweeps of 10° wedges were collected from 9 CrystalDirect loops, out of which 375 datasets were successfully processed. 101 datasets were selected based on Δ -CC 1/2 (Assmann et al, 2020) and ISa values as implemented in sxdm tool (Basu et al, 2019), followed by merging with XSCALE. The final mtz file was used for structure determination in Molecular replacement method.…”