The crystal loop detection algorithm is based on finding out the crystal loop ending point opposite to the crystal loop pin using image cross section digital image column profile analysi
Trang 1Physics Procedia 84 ( 2016 ) 315 – 320
1875-3892 © 2016 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license
( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
Peer-review under responsibility of the organizing committee of SFR-2016.
doi: 10.1016/j.phpro.2016.11.053
ScienceDirect
International Conference "Synchrotron and Free electron laser Radiation: generation and
application", SFR-2016, 4-8 July 2016, Novosibirsk, Russia Some aspects of crystal centering during X-ray high-throughput
protein crystallography experiment Yu.A.Gaponov a,*, N.Matsugaki b, K.Sasajima b, N.Igarashi b, S.Wakatsuki c
a Department of atmospheric research, Novosibirsk State University, Pirogova 2, Novosibirsk, 630090, Russia
b Structural Biology Research Center, High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, 305-0801, Japan
c Stanford University, Stanford, California, 94305, USA
Abstract
A set of algorithms and procedures of a crystal loop centering during X-ray high-throughput protein crystallography experiment has been designed and developed A simple algorithm of the crystal loop detection and preliminary recognition has been designed and developed The crystal loop detection algorithm is based on finding out the crystal loop ending point (opposite to the crystal loop pin) using image cross section (digital image column) profile analysis The crystal loop preliminary recognition procedure is based on finding out the crystal loop sizes and position using image cross section profile analysis The crystal loop fine recognition procedure based on Hooke-Jeeves pattern search method with an ellipse as a fitting pattern has been designed and developed The procedure of restoring missing coordinate of the crystal loop is described Based on developed algorithms and procedures the optimal auto-centering procedure has been designed and developed A procedure of optimal manual crystal centering (Two Clicks Procedure) has been designed and developed Developed procedures have been integrated into control software system PCCS installed at crystallography beamlines Photon Factory BL5A and PF-AR NW12, KEK
© 2016 The Authors Published by Elsevier B.V
Peer-review under responsibility of the organizing committee of SFR-2016
Keywords: X-ray crystallography; high-throughput protein crystallography; control system; SR; object detection and recognition; Linux
* Corresponding author Tel.: +7-913-065-8195
E-mail address: yurii3000@gmail.com
© 2016 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license
( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
Peer-review under responsibility of the organizing committee of SFR-2016.
Trang 21 Introduction
Crystal centering procedure is important part of X-ray protein crystallography experiment A typical protein crystal holder is tiny nylon loop with a size about 50÷500μm A typical protein crystal size is about 40÷400μm It takes several minutes to center manually protein crystal under investigation after mounting crystal holder onto X-ray beam area In contrast, to center the crystal automatically during the X-ray high-throughput protein crystallography experiment becomes not simple procedure There are several steps of centering the crystal as it was described by Karain et al (2002) and Pauluhn et al (2011) Steps typically include several consequent detections, recognitions, rotations around goniometer axis and shifts along coordinate axes using data analysis of the X-ray beam area optical images with different magnification of monitoring optical system In the case, when the crystal size is about the crystal loop size, centering procedure is completed after centering the crystal loop In the case, the crystal is smaller than the crystal loop, crystal centering procedure is started, which is similar to crystal loop centering procedure but more complicated and less reliable
The step of detection and recognition of the crystal loop and the crystal in the loop is crucial in the centering procedure There are several approaches to solve the problem One of it, as it was described by Roth et al (2002) and Jain et al (2007), is image analysis using digital filters to reduce the image noise and to determine image pixel’s intensity gradient or standard deviation of the intensity to detect crystal loop, crystal edges and crystal corners Another approach, as it was described by Pauluhn et al (2011), is calculation of ratio of black and white pixels of preliminary converted image onto black and white mode to find the boundaries of the crystal loop Nevertheless all algorithms have some percentage of fault cases of centering and the problem of crystal loop and crystal centering still is actual
In this report we describe simple algorithm of crystal loop detection and preliminary recognition based on profile analysis of optical image cross-section curves A procedure of crystal loop fine recognition using Hooke-Jeeves pattern search method with an ellipse as a fitting pattern has been designed and developed An optimal manual crystal centering procedure (Two Clicks Procedure) is described
2 Crystal loop centering
Schematically the optical image of the X-ray beam area is defined as shown at Fig.1a The goniometer is on the
left side of the optical image of X-ray beam area Goniometer axis G axis coincides with axis X of optical image of the
X-ray beam area and crosses X-ray beam Coordinate axes X and Y of the X-ray beam area coincides with coordinate axes X and Y of the optical image Coordinate origin of the X-ray beam area coincides with coordinate
origin of the optical image and is in the center of the optical image The X-ray beam crosses coordinate origin of the optical image
Crystal loop centering algorithm is based on finding coordinates (x cp , y cp , z cp) of some centering point of the
crystal loop Two coordinates (x cp , y cp) can be obtained using the optical image of the X-ray beam area directly by clicking the mouse pointer onto the centering point of the crystal loop (manual centering mode) or automatically
(auto-centering mode) Unknown centering point coordinate z c can be obtained after solving the geometric task with
a set of two XY point coordinates before and after rotation the crystal loop by angle α=90° around the axis X using
right screw along the positive direction of the axis X, as it is described in Korn and Korn (1968):
൭
ݔ
ݕ
ݖ൱ ൌ ൭
Ͳ ܿݏ ߙ െ ݏ݅݊ ߙ
Ͳ ݏ݅݊ ߙ ܿݏ ߙ ൱ ൭
ݔ
ݕ
ݖ൱ ൌ ൭
ݔ
െݖ
where (x cp , y cp , z cp ), (x cpr , y cpr , z cpr) – centering point coordinates before and after the crystal loop rotation Other two
coordinates (x cpr , y cpr ) are obtained in the same manner as coordinates (x cp , y cp) at previous step The unknown
coordinate z c is defined from (2):
Trang 3At the next step the goniometer XYZ shifting mechanism shifts the crystal loop into the X-ray beam position
(0, 0, 0) with shift vector r sh =(-x cp , -y cp , -z cp)
Fig 1 The steps of crystal loop detection and crystal loop preliminary recognition: (a) the optical image of the X-ray beam area schematic view,
(b) the step of crystal loop ending point detection, (c) the step of crystal loop preliminary recognition
3 Crystal loop detection
The noise of the X-ray beam area optical image is reduced by convolution of the image pixels with a simple matrix:
K=ͲǤͳ ͲǤͳ ͲǤͳͲǤͳ ͲǤʹ ͲǤͳ
ͲǤͳ ͲǤͳ ͲǤͳ
It is assumed that the optical image mode is grayscale one Typically, crystal loop intensity is lower than the intensity of the image background In this case, the pixel’s intensity of optical image is inverted according to relation
I ij inv = I ij max - I ij ǡwhere I ij max – the biggest value of pixel’s intensity of the image (see Fig.1a, b)
It is assumed that at right side of the image nothing should be observed except crystal loop oriented
approximately along axis X That is why crystal loop detection starts finding out the crystal loop from the right side
of the image (the opposite side to the crystal pin side, which is on the left) The analysis starts from the right side of the optical image, taken at lowest possible magnification of optical system after mounting the crystal loop at the goniometer
Trang 4Crystal loop ending point detection algorithm is based on analysis of optical image columns one by one starting
from the right side of the optical image The analysis stops when a profile with some intensity I e will be recognized
at some coordinate y e That assumed to be the crystal loop ending point (see Fig.1b) with coordinates (x e , y e) Then
using the goniometer XYZ shifting mechanism the crystal loop is shifted into the center of optical image position
(0, 0) using shift vector r sh =(-x e , -y e) The step of finding the crystal loop ending point is repeated until the physical crystal loop ending point will be find out (just after mounting the crystal loop might be partly out of the optical image) and moved into the center of optical image
Finally, the crystal loop ending point is centered with use of the crystal loop centering algorithm After that, the crystal loop ending point is assumed to be mounted into the X-ray beam position
4 Crystal loop recognition
At the step of crystal loop preliminary recognition, the sizes and position of gravity center of the crystal loop are determined The image columns are analyzed to calculate the crystal loop ݕ-width within the crystal loop ending
point (x e , y e) of the crystal loop ending point For this purpose, intensity of pixels in column is checked from upper
side of the image to the bottom Checking is stopped at some coordinate y upp, when the intensity of the pixel will be
within the registered at previous step intensity I e Then the intensity of pixels in the same column is checked from
bottom side of the image to upper Checking is stopped at some coordinate y low, when the intensity of the pixel will
be within the registered at previous step intensity I e The crystal loop y width is defined as y width = (y upp - y low) The
crystal loop center position Y-coordinate y cp is defined as y cp = (y upp + y low )/2 The crystal loop x width is defined as
x width ≈ -2x cur , where x cur is the current coordinate of image column under analysis The crystal loop center position
X-coordinate x cp is defined as x cp ≈ -x width The image columns are analyzed until the value of y width stops to increase
within the condition x width <10y width in the case when y width is not changed Typically, the value of y width stops to increase within the crystal loop center Then the crystal loop is rotated with 45° several times (up to 4) to define the
biggest value of y-width repeating the described step The angle position of goniometer Ω cp with the biggest value of
y-width is angle at which crystal loop is approximately parallel to the optical image plane After determining the
angle position of goniometer Ω cp , crystal loop is rotated onto angle position Ω l-45° Then the crystal loop center
point (x cp , y cp) is centered using the crystal loop centering algorithm After that, the crystal loop center point is assumed to be mounted into the X-ray beam position
The final step of crystal loop recognition is based on the fitting the crystal loop with a predefined shape using Hooke-Jeeves pattern search method, which was described by Hooke et al (1968) An ellipse pattern shape fits the crystal loop square within 10÷20% and improves results of the crystal loop preliminary recognition algorithm described above At first, the ellipse center is placed into the crystal loop center position calculated at previous step
Then intensity of all pixels covered by the ellipse are integrated resulting in value I ellipse Then parameters of the
ellipse consequently are changed by some value followed by recalculation of the value I ellipse In the case, when the
value of I ellipse decreases ellipse parameter changing is rejected After checking all ellipse parameters the changing value is decreased by factor 2 and fitting process is repeated The crystal loop recognition process is stopped when values of parameter changing becomes smaller than minimal values, which depends on sizes of ellipse The position
of the ellipse is used as a crystal loop center during the final crystal loop centering
5 Beamline control software
All described algorithms and procedures where integrated into beamline control software system PCCS, which was described by Gaponov et al (2004) at crystallography beamlines Photon Factory BL5A and PF-AR NW12, KEK Fig.2 shows the PCCTools application with crystal centering mode of operating For manual crystal centering, the optimal procedure (Two Clicks Procedure) was realized: one mouse right button click on the crystal under investigation (after mounting the crystal loop at the goniometer) activates crystal centering procedure Click coordinates are stored and then goniometer rotates crystal loop by angle 90° The second mouse right button click on the crystal activates crystal centering procedure The automatic crystal loop centering procedure is activated by
Trang 5clicking the button “Centering” The automatic crystal loop centering procedure is used in high throughput mode just after mounting at goniometer the next crystal loop by sample robot changer
Fig 2 The PCCTools application with crystal centering mode of operating
6 Conclusion
To optimize the processing time and reliability of crystal loop centering a simple procedure of the crystal loop detection and preliminary recognition in predefined geometry of the goniometer X-ray beam area has been designed and developed In result, the procedure finds out the sizes of the crystal loop, calculates the center of found out shape and moves the found out center point into the beam position
To increase the accuracy of the crystal loop centering a fine recognition procedure based on Hooke-Jeeves pattern search method with an ellipse as a fitting pattern has been designed and developed In result, the procedure finds out the center of the crystal loop (considered as an ellipse) and moves the crystal loop center point into the beam position
A procedure of optimal manual crystal centering (Two Clicks Procedure) has been designed and developed
All procedures for crystal loop and crystal centering have been integrated into control software system PCCS installed and commissioned at crystallography beamlines Photon Factory BL5A and PF-AR NW12, KEK
Acknowledgements
The authors would like to appreciate M.Hiraki for valuable and constructive discussions about crystal centering problem Special thanks to K.Demura for help during the beamline control software installation
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, Special Coordination Funds for Promoting Science and Technology, and the Protein 3000 Project of the MEXT
Trang 6References
Karain, W., I., Bourenkov, G., P., Blumea, H., Bartunika, H., D., 2002 Automated mounting, centering and screening of crystals for high-throughput protein crystallography Acta Cryst D58, 1519-1522
Pauluhn, A., Pradervand, C., Rosetti, D., Salathe, M., Shulze-Briese, C., 2011 Automatic loop centering with a high-precision goniometer head
at the SLS macromolecular crystallography beamlines J Synchrotron Rad 18, 595-600
Roth, M., Carpentier, P., Kaȉkati, O., Joly, J., Charrault, P., Pirocchi, M., Kahn, R., Fanchon, E., Jacquamet, L., Borel, F., Bertoni, A., Israel-Gouy, P., Ferrer, J.-L., 2002 Acta Cryst D58, 805-814
Jain, A., Stojanoff, V., 2007 Are you centered? An automatic crystal-centering method for high-throughput macromolecular crystallography J Synchrotron Rad 14, 355–360
Korn, G., A., Korn, T., M., 1968 Mathematical handbook for scientists and engineers McGraw-Hill, New York, pp 1130
Hooke, R., Jeeves, T., A., 1961 "Direct Search" solution of numerical and statistical problems J ACM 8-2, 212-229
Gaponov, Yu., A., Igarashi, N., Hiraki, M., Sasajima, K., Matsugaki, N., Honda, N., Yamada, Y., Wakatsuki, S., 2004 Secure UNIX socket-based controlling system for high-throughput protein crystallography experiments, J Synchrotron Rad 11, 17-20