Ezequiel Panepucci

Fast native-SAD phasing for routine macromolecular structure determination

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

In meso in situ serial X-ray crystallography of soluble and membrane proteins

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase is described. It works with microgram quantities of protein and lipid (and ligand when present) and is compatible with the most demanding sulfur SAD phasing.

In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures.

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase at cryogenic temperatures (100 K) is described. It works with nanogram to single-digit microgram quantities of protein and lipid (and ligand when present), and is compatible with both high-resolution native data collection and experimental phasing without the need for crystal harvesting.

PRIGo: a new multi-axis goniometer for macromolecular crystallography

The design and performance of the new multi-axis goniometer PRIGo developed at the Swiss Light Source at Paul Scherrer Institute is described.

Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons.

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000–10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.

EIGER detector: application in macromolecular crystallography

The application of the EIGER hybrid photon-counting pixel detector in macromolecular crystallography is presented. Data-collection strategies exploiting the unique features of EIGER are discussed.

D3, the new diffractometer for the macromolecular crystallography beamlines of the Swiss Light Source

A new diffractometer for microcrystallography has been developed for the three macromolecular crystallography beamlines of the Swiss Light Source.

Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing protein crystals

This article reports the incorporation of a fast continuous grid scan with both still and oscillation images into the Swiss Light Source macromolecular crystallography beamlines and its application in visualization of protein crystals with scanning transmission X-ray microscopy.

DA+ data acquisition and analysis software at the Swiss Light Source macromolecular crystallography beamlines

The implementation of DA+ data acquisition and analysis software at Swiss Light Source macromolecular crystallography beamlines is presented. Detailed descriptions of the user interface, acquisition engine, online processing and database, which are the major components of DA+, are given.

Fast and accurate data collection for macromolecular crystallography using the JUNGFRAU detector

The accuracy of X-ray diffraction data is directly related to how the X-ray detector records photons. Here we describe the application of a direct-detection charge-integrating pixel-array detector (JUNGFRAU) in macromolecular crystallography (MX). JUNGFRAU features a uniform response on the subpixel level, linear behavior toward high photon rates, and low-noise performance across the whole dynamic range. We demonstrate that these features allow accurate MX data to be recorded at unprecedented speed. We also demonstrate improvements over previous-generation detectors in terms of data quality, using native single-wavelength anomalous diffraction (SAD) phasing, for thaumatin, lysozyme, and aminopeptidase N. Our results suggest that the JUNGFRAU detector will substantially improve the performance of synchrotron MX beamlines and equip them for future synchrotron light sources.A charge-integrating pixel-array detector called JUNGFRAU enables the collection of highly accurate X-ray crystallography data at synchrotron sources at unprecedented speeds.