Cheiron School BL Practice of BL38B1 (Protein Crystallography)
Title:
Data Collection and S-SAD Phasing of Insulin and Lysozyme Crystals
Staff:
Kazuya Hasegawa, Seiki Baba
(Structural Biology Group, SPring-8/JASRI)
Abstract:
Phase problem is a major difficulty in protein crystallography. As a recent advancement, S-SAD
phasing method has been proposed. It solves the problem by the anomalous effect of sulfur
atoms involved in most protein samples. In this exercise, we plan to the data collection ofinsulin crystal and the determination of its structure. The alignment of the beamline and asample mounting robot will also be introduced.
Contents:1.

Brief Introduction
Energy range 6 ∼ 17.5 keVEnergy resolution ΔE/E = 10-4
Spot size horizontally 87µm, vertically 142 µm (@ 12.4 keV (λ = 1.0 Å)) at sample
position.

Fig 1. BL38B1. (Left) Inside of experimental hutch. (Right) Outside. Operators desks andcomputers are located.

1.1.

Optical Components
Simple optics which consists of SPring-8 standard double crystal monochrometor and2-dimensional focusing bend cylindrical mirror is adopted.

Fig. 2: Schematic View of Beamline

Fig. 3: Outline of Optics
1.2.

Experimental Station
Rapid data collection system for protein crystallography is installed at BL38B1.
Combination of the coaxial-microscope to observe crystal along x-ray beam path and thegoniometer having motor driven x, y and z stage make centering of crystal very easy.
Rapid readout CCD detector with a detective area of 315×315mm2 enables efficient datacollection with minimum dead time and high resolution data collection even when using
long wavelength.
Data collection, including measurement of XAFS spectrum and automatic MAD(Multi-wavelength Anomalous Diffraction) data collection, is performed throughcontrolling software BSS (Beamline Scheduling Software). Since BSS can control all of
the instruments required for data collection, switch of detector and change of camera
length, wavelength are automatically performed at the beginning of measurement.
Therefore, what beamline users have to do after crystal mounting and centering is to input
measurement conditions to BSS.

Fig. 4. Diffractometer at BL38B1

Fig.5. Magnified view around the goniometercoaxial-microscope with a zoom lens enables easy crystal centering1.3.

Specification of beamline equipments* CCD detector (ADSC/ Quantum315)
Detector area : 315 × 315 mm2Pixel size : 51 × 51 µm2 (unbin)
No. of pixels : 6144 × 6144
Camera length : 75 ∼ 950 mm* Horizontal axis goniometer having motor-driven x, y, z stage* Cryostat : temperature control range 80 ∼ 350 K* Gas-flow type ionization chamber* Si PIN photodiode detector for fluorescence measurement* Multi-channel analyzer of fluorescence measurement* Control Software BSS* Sample Automated Changer

Fig. 6: Sample Auto Change SPACE2.

Optical Alignment and Adjustment

2.1.

Control Software BSS
The software of BSS (Beamline scheduling software) can deal with all of the beamline
operation as follows: 1) Control the optical and experimental components as
monochromator, goniometer, and detector. 2) Data collection of crystal diffraction, X-ray
fluorescence etc.
The GUIs of the software is as follows:

Fig. 7: BSS Crystal Centering Tab

Fig. 8: BSS Device Control Tab
2.2.

Operations of Beam Alignment

2.2.1.

Confirm and define the rotational center of the goniometer head at the window of thesample monitor software using a sample mount Lithographic loop. If the position is not
located at the center of the display, the position should be aligned by the micrometer of thesample monitor CCD camera. The cross of the center position can also be aligned by thesoftware definition of the video capture software.

2.2.2.

BSS Run Option is set to ”Admin” mode.

2.2.3.

Set the spindle axis to 0 degree.

2.2.4.

Put the fluorescent plate on the goniometer head.

2.2.5.

Focus the microscope on the surface of the fluorescent plate by moving stl_gonio_1_x axis
from Axis Tools window of BSS.

2.2.6.

Turn off the sample illuminator.

2.2.7.

Set wavelength 1 Å and open X-ray shutter.

2.2.8.

Record the X-ray intensity at 1A (Electric current of ionization chamber).

2.2.9.

Display the monochromator alignment GUI on BL-Work Station. Record the position of Δθ1axis.

2.2.10. The size of two slits (ST1/ST2) is set to 3 mm by Axis Tools.
2.2.11. Tune Δθ1 to maximize intensity by using the Energy tab of Axis Tools. Record the I0 value,the position of the beam, and the shift of Δθ1.
2.2.12. Beam position should be aligned to the rotational center of the goniometer by the translating
the experimental stage. In Z axis direction (height), beam position should be placed on thespindle axis. In Y axis direction (horizontal), the position should be the central cross of thevideo monitor.
2.2.13. Change the slit aperture size to the original values. The aperture size of ST1 and ST2 are
0.5/0.3mm and 0.4/0.4mm, respectively. Record the I0 value.
2.2.14. Tune Δθ1 again.
2.2.15. BSS Run Option is set to ”None” mode.

3.

Data Collection and Phasing of Insulin Crystals

3.1.

Theoretical background of SAD phasing

3.1.1. Anomalous effectThomson scattering occurs the following scheme: 1) X-ray photon strikes a freeelectron, 2) the electromagnetic field of the incident oscillates the electron. 3) theoscillated electron scatters X-ray with the same wavelength as the incident X-ray.
However, the electrons of atoms are bound tightly and its oscillation has a certain
resonance frequency. Therefore, the scattering factor is depends on the incident
wavelength. It can be expressed as the following formula.

f = f o + f '+if "In the case of sulfur atom, the wavelength dependent f’ and f” terms are illustrated asfollowing graphs.

Fig. 9. Anomalous scattering terms of sulfur atom. Calculated by CCP4/Crossec.
3.1.2. Harker diagram and phase
Crystallographic F(h) values (structure factors) are given by Fourier transformation ofelectron density ρ(x). F(h) value is a complex and composed of absolute |F(h)| andphase a(h). Inverse Fourier transformation of F(h) gives electron density of crystal,
however |F(h)| is only obtained from diffraction experiments. The phase a(h) can berecovered from several experimental methods. Here we describe the scheme of SAD
method.
Harker diagram (phase diagram) is a picture for understanding phase determination.

Fig 10: Harker diagram of SAD method.
Anomalous effect breaks down Friedel’s Law where a reflection F(h) and its
centrosymmetric reflection F(-h) have same value of |F|. ΔF, the difference of F(h) andF(-h) is a good approximation of the structure factor amplitude of anomalous scatterers.
Therefore, Patterson method or direct phasing can extract the position of the anomalousscatterers.
Once the positions of anomalous scatterers are determined, their structure factor (F”H) iscalculated. The vector of F”H and its negative can be drawn in the diagram. Next, we
can draw two circles: one with radius of |F(+)P| centered on the end of F”H. another withradius |F(-)P| centered on the end of –F”H. We can see the two intersections FP and FP~ ofthe two circles. One of the two points is correct answer. Although the two possibilities
cannot be distinguished without any information, we can obtain good phases by thetreatment of electron density shape (density modification; solvent flattening etc.) insome cases.

3.2.

Data Collection

3.2.1. Sample mounting on goniometer
1) Pick up a desired crystal by a cryoloop device: Firstly, a desired crystal is scoopedwith a cryoloop under a microscope. Protein crystal is mostly small and its typicaldimension of 0.1~0.2 mm. Protein crystal is obtained from water solution, therefore it iseasy to dry up under atmospheric condition and also very fragile. Scooping process
should be conducted rapidly.

Fig. 10. Cryoloop. (Left) Cryoloop, (Middle) Nylon loop. The tip of the cryoloop.
(Right) Cryoloop with a crystal.
2) Soaking the crystal into cryoprotectant: The scooped crystal is transferred into thesolution containing cryoprotectant (typically it contains 30% glycerol) which prevents
ice formation during sample cooling.
3) Picking the crystal again: To mount the crystal onto a goniometer head, the crystal ispicked again. Since glycerol containing solutions are viscous, the excess of the solvent
is sometime scooped onto the cryoloop. The excess causes unfavorable scattering which
degrades the statistics of diffraction data. Since the anomalous signal for S-SAD
phasing is weak, the solvent should be removed possibly.
4) Mounting and flush cooling the crystal: Immediately, the picked crystal should be puton the goniometer head with magnetic support. To cool the sample during X-ray
irradiation and prevent the radiation damage of the sample, cold nitrogen gas stream isblowing at the crystal position. Before mounting the crystal, the blow is blocked out by
the shutter system. And after mounting, the shutter should be immediately opened. Thisis ‘flush cooling technique’ for preventing ice formation in solvent and crystal.
3.2.2. Collection of diffraction images
1) Set up the data collection software: To take diffraction images, the program BSS isused at this beamline. Following two figures are the windows of data collection GUI.

Fig. 11: Main window of BSS

Fig. 12: The window for editing data collection condition.
2) Centering the sample to beam position: Diffraction images are taken by oscillation

method. In the method, crystal rotates and oscillates with the spindle axis of goniometer.
To keep the crystal position during the rotation against X-ray beam, the crystal isaligned to the center of the spindle axis, because X-ray beam is already aligned to thespindle axis. This operation can be achieved in the program BSS using video cameraand monitor.
3) Closing experimental hutch and opening down stream shutter of the beamline.
4) Take a few images and check the diffraction: To estimate the quality of the mounted
crystal, one or two images should be taken before taking data set.
5) Take a full data set: When the crystal passes through the quality check, theconsecutive series of diffraction images are taken. In most cases, 180 images will betaken if you do not have any information of the crystal as space group, cell constant etc.
In the case of SAD, highly redundant data collection is required, because it makes thestatistics better.

Fig. 13: A diffraction pattern of a protein crystal.
3.3.

Data processing
Diffraction images are processed by a special computer program. Against the raw data ofeach image, the following process is performed: 1) the prediction of reflections(diffraction spots) through the determination of lattice constants and crystal orientationand the parameter-refinement of the camera-distance etc [indexing and prediction]. 2) thedetermination of reflection index and the estimation of its intensity [integration]

Fig: 14: Spot prediction

After obtaining the index and intensity of each reflection in all images, the intensity
should be merged into reflections which are equivalent in crystallographic symmetry[scaling]. Finally we obtain F-data (containing hkl: Mirror index and F(hkl): structure
factor amplitude). In the case of SAD, Friedel mates F(+) and F(-) should be recorded
independently.
3.4.

Phase Calculation by SAD
There are several program packages for this purpose. In this practice, HKL2MAP will beused. This program is a GUI software for SHELX crystallographic analysis package. This
analysis is performed as following procedure: 1) SHELXC: the preparation of ΔF datafrom experimental F data. 2) SHELXD: finding the positions of sufur atoms asanomalous scatterers. 3) SHELXE: phase calculation and phase improvement by density
modification. Finally, the calculated phases are checked by electron density maps using
the program XtalView/Xfit. Its details will be introduced at the BL practice. Only several
pictures are shown.

Fig. 15: hkl2map (Shelxc)

Fig. 16: hkl2map (Shelxd)

Fig. 17: CCall vs. CCweak

Fig. 18: Histogram CCall

Fig. 19: Estimate Solvent Content. Insulin is composed of 51 amino acids

Fig. 20: Shelxe

Fig 21: Contrast graph.

Fig. 22: Nicely determined phases. High contrast electron density map was obtained.