NSLS-II biomedical beamlines for micro-crystallography, FMX, and for highly automated crystallography, AMX: New opportunities for advanced data collection

Zhu

et al. 2021

Preprint

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Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a dominant phasing method for the crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database for finding suitable templates for phasing. However, such sequence information is not always available such as when proteins are crystallized with unknown binding partner proteins or when the crystal is that of a contaminant. The recent development of AlphaFold has resulted in the availability of predicted protein structures for all proteins from twenty species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough for sequence-independent phasing of diffraction data from two crystallization contaminants for which we had not identified the protein. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained which allowed the identification and structure determination of both structures, YncE and YadF. Our results advocate a general utility of AlphaFold-predicted structure database with respect to crystallographic phasing.

“…Diffraction data were collected at the NSLS-II beamline FMX (17ID-2) at 100 K (Fuchs et al ., 2016, Schneider et al ., 2021). The beamline is equipped with an Eiger 16M detector.…”

Section: Methodsmentioning

confidence: 99%

AlphaFold Protein Structure Database for Sequence-Independent Molecular Replacement

Zhu

et al. 2021

Preprint

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“…Crystals were cryo-protected in 0.8 M NaH 2 PO 4 , 1.2 M K 2 HPO 4 , 0.2 M NaCl, and 20% glycerol and flash-frozen in liquid nitrogen. Diffraction data was collected at 100 K using an Eiger 9M pixel-array detector on beamline 17-ID-1 (AMX) at Brookhaven National Laboratory [70,71].…”

Section: Crystallization and Structure Determinationmentioning

confidence: 99%

Influence of the amino-terminal sequence on the structure and function of HIV integrase

et al. 2020

Background: Antiretroviral therapy (ART) can mitigate the morbidity and mortality caused by the human immunodeficiency virus (HIV). Successful development of ART can be accelerated by accurate structural and biochemical data on targets and their responses to inhibitors. One important ART target, HIV integrase (IN), has historically been studied in vitro in a modified form adapted to bacterial overexpression, with a methionine or a longer fusion protein sequence at the N-terminus. In contrast, IN present in viral particles is produced by proteolytic cleavage of the Pol polyprotein, which leaves a phenylalanine at the N-terminus (IN 1F). Inspection of available structures suggested that added residues on the N-terminus might disrupt proper protein folding and formation of multimeric complexes. Results: We purified HIV-1 IN 1F 1-212 and solved its structure at 2.4 Å resolution, which showed extension of an N-terminal helix compared to the published structure of IN 1-212. Full-length IN 1F showed increased in vitro catalytic activity in assays of coupled joining of the two viral DNA ends compared to two IN variants containing additional N-terminal residues. IN 1F was also altered in its sensitivity to inhibitors, showing decreased sensitivity to the strandtransfer inhibitor raltegravir and increased sensitivity to allosteric integrase inhibitors. In solution, IN 1F exists as monomers and dimers, in contrast to other IN preparations which exist as higher-order oligomers. Conclusions: The structural, biochemical, and biophysical characterization of IN 1F reveals the conformation of the native HIV-1 IN N-terminus and accompanying unique biochemical and biophysical properties. IN 1F thus represents an improved reagent for use in integration reactions in vitro and the development of antiretroviral agents.

“…The two recently opened beamlines for macromolecular crystallography at NSLS-II have launched the microcrystallography field into new territory, with flux densities up to two orders of magnitude brighter than those previously available [3]. The Frontier Microfocusing Macromolecular Crystallography beamline (FMX, X17-ID-2) has a beam size of 1 × 1.5 µm, expandable to 10 µm and a flux of 3.5 × 10 12 photons/s.…”

Section: Introductionmentioning

confidence: 99%

“…The Frontier Microfocusing Macromolecular Crystallography beamline (FMX, X17-ID-2) has a beam size of 1 × 1.5 µm, expandable to 10 µm and a flux of 3.5 × 10 12 photons/s. The Highly Automated Macromolecular Crystallography beamline (AMX, X17-ID-1) has a beam size of 5 × 7.5 µm and a flux up to 5 × 10 12 photons/s [3]. This state-of-the-art technology unlocks new possibilities for microcrystal diffraction and serial crystallography experiments.…”

Section: Introductionmentioning

confidence: 99%

Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II

et al. 2019

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Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II—AMX and FMX—deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins—Akt1, PI3Kα, and CDP-Chase—to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or “raster-collect”, a more automated approach for a larger number of crystals (using CDP-Chase as an example).