Photon Factory Activity Report 2016 #34 (2017)

BL-1A/2016G664

Strategies for native-SAD experiment at low X-ray energy: Users’ Reports
Shibom Basu1, Vincent Olieric1, Tomizaki Takashi1, Chia-Ying Huang1, Justyna Wojdyla1, Naohiro
Matsugaki2, Meitian Wang1,*1

Swiss Light Source, Paul Scherrer Institut, Villigen-5232, Switzerland
Structural Biology Research Center, Photon Factory, High Energy Accelerator ResearchOrganization, Tsukuba, Japan2

1 Introduction
Native-SAD(Single-wavelength
AnomalousDiffraction) phasing is becoming the dominant methodtowards de-novo X-ray structure determination ofbiomolecules. This method relies on weak anomalous
intensities from the light anomalous scatterers (e.g. S or P
atoms or ions natively present in biomolecules). Theselight elements have X-ray absorption edges in the regionof 2-3 keV, which cannot be reached at mostmacromolecular crystallography beamlines. However,
BL1A beamline at Photon Factory has a unique setup,
where low X-ray energy can be achieved down to 3.7
keV. One of the major difficulties of native-SAD
experiments at low X-ray energy is the high absorption.
Here, long absorption X-ray path length is detrimental tothe anomalous signal from S atoms. Presence of solventin the loop, cryo-stream of liquid N 2 and air around thecrystal can further outweigh the gain in anomalous
signals. However, the diffraction efficiency of crystals issupposed to increase in the order of square of λ. It has
been noticed that for crystal sizes smaller than 100-μm,
use of low X-ray energy can increase the anomalous
signal from S atoms. Fig. 1a shows the calculated
anomalous signals for different crystal size and X-ray
wavelengths, in absence of any absorption effect.
In this work, we performed native-SAD measurement
on crystals of various thicknesses with aim at improving
data quality by minimizing the X-ray absorption effect at
low X-ray energy.
2 Experimental setup and Data collection
We devised an experiment, where we tried to achieve
the ideal sample preparation for experiment at lowenergy. We used lysozyme crystals of various sizes
mounted on Micro-acti-loop from MiTeGen with reduced
amount of solvent. The crystals were mounted on agoniometer, cryo-cooled and collected at 4.59 and 3.75
keV X-ray energies.
For native-SAD measurement, we first normalized thetransmissions of different X-ray wavelengths (2.7 and 3.3Å) using a long 50 µm shaped lysozyme crystal. A Vshaped detector configuration with two EIGER X 4M
detectors was used with a tilt angle of 25° (See Fig. 1b) inorder to catch high-angle reflections at 3.3 Å (3.75 keV).
We used 50x50 µm beam. BL-1A is equipped with a Hechamber to reduce air absorption. We used 3.04% and2.27% transmissions at 2.7 and 3.3 Å X-ray wavelengths

respectively. Datasets were collected with oscillation
wedge of 0.2° and exposure time of 0.1 sec.
3 Results and Discussion
Successful native-SAD measurement implies high data
quality and high anomalous signal while keeping highangle reflections. The V-shape detector configuration
provided highest resolution of 2.4 Å compared to the flat
configuration which could provide resolution only up to3.4 Å at 3.75 keV. This improvement in resolution was
important for successful substructure determination at 3.3
Å X-ray wavelength (3.75 keV energy). BL1A
experimental setup allows the reduction of absorption
effects at low X-ray energy. So, comparison of anomaloussignals (Fig. 1c) attained at 2.7 Å (4.59 keV) and 3.3 Å(3.75 keV) X-ray wavelengths shows that longer
wavelength native-SAD could indeed produce higher
anomalous signals. As a result, gain in anomalous signals
could be utilized for successful phasing of lysozyme andstructure could be determined at 3.75 keV (Fig. 1d). This
study helped us to understand optimum crystal size forlong-wavelength data collection.
After successfully establishing the strategy withlysozyme crystals, we want to apply it to real-life
challenging proteins crystals.

Fig 1. Ideal native-SAD measurement, experimental
setup, and electron density at 3.3 Å (or, 3.75 keV) Xray wavelength. 1a. It shows a 2-D contour plot for
optimal anomalous intensity for a given crystal size and
X-ray wavelength in ideal condition (i.e., no absorption
effect) based on a simulation. 1b. It represents the Vdetector configuration at BL-1A beamline andPhoton Factory Activity Report 2016 #34 (2017)

experimental setup. 1c. It compares the anomalous signals
between two X-ray wavelengths – 2.7 and 3.3 Å based ondata collected on the same 50 µm shaped crystal. 1d. The
electron density of lysozyme crystal contoured at 1.5 σ,proving successful native-SAD structure determination at
3.3 Å X-ray wavelength.
Acknowledgement
The project was funded by Paul Scherrer Institut,Switzerland. We are thankful to BL-1A staff at Photon
Factory, KEK in Japan.
References[1] Publication in preparation* meitian.wang@psi.ch