For preparation of ZOO sample sheet
as of 2020-12-15<Reference of automatic data collection and automatic data processing>If you publish your structures from data collected using ZOO and analyzed withKAMO, please cite the following papers as references. The citations areexceedingly important for us to continue developing more advanced techniquesat synchrotron radiation technology.
Thank you for your cooperation.
All staff
1. K. Hirata, K. Yamashita, G. Ueno, Y. Kawano, K. Hasegawa, T. Kumasaka,and M. Yamamoto
ZOO: an automatic data-collection system for high-throughput structure
analysis in protein microcrystallography
Acta Cryst. D75, 1–13, (2019).
[doi: 10.1107 / S2059798318017795]
2. K. Yamashita, K. Hirata, and M. Yamamoto
KAMO: towards automated data processing for microcrystals
Acta Cryst. D74, 441-449, (2018).
[doi: 10.1107 / S2059798318004576]<Automated data collection sample sheet>
When executing automated data collection, the measurement conditions must
be described on the automated data collection sample sheet (hereafter“sample sheet”).
Download and edit the sample sheet and send it to the contact address (mailin@spring8.or.jp) by the specified date.
If you have any questions or concerns, please feel free to ask us via the contactaddress.
<Naming rule for the sample sheet>
Please follow the following naming rules.
File name should be
ZOOPREP_YYMMDD_NAME_BLNAME.xlsx (valid as of 2020-09-28)
YYMMDD:
Date (entry date of the sample sheet) 2019-10-02 -> 191002
NAME:
Enter the name of the experimenter. Please capitalize the first letter of your firstname and that of your last name with no space between. The name of yourinstitution/organization is also acceptable. Do not include "_" in your name.
1Good example: ZOOPREP_191002_KunioHirata_BL32XU.xlsx
Bad example: ZOOPREP_191002_Kunio_Hirata_BL32XU.xlsx
BLNAME:
BL32XU or BL45XU or BL41XU<Outline of ZOO measurement>
For each measurement scheme shown below, ZOO can be conductedautomatically.

(small wedge = multi mode)<Keep in mind>
Automated data collection is executed under the conditions described in thesample sheet from top to bottom.
Please consider your preferred priorities and input them on the sheet.
ZOO cannot currently do the following (but are being considered forimplementation).
✔ Executing data collection when the resolution is higher than given criteria atraster scan.
✔ Intelligent skipping: Skip residual sample pins when it can acquire one or two
good datasets.
✔ Snapshot at 0º and 90º2

<Parameters of the automatic measurement sample sheet>
The following is an explanation of each parameter.
●PuckID:■ ID of UniPuck■ Enter the ID stamped on the UniPuck lid (SPring-8 definition: ‘lid’ hasmore metal, thus heavier, than the bottom "Sample enclosure")■ When BL staff set the UniPucks into the sample changer, this parameter
will be checked against the IDs on their lids.
■ DO NOT use hyphens for this parameter, ‘PuckID’, on the sample sheet●PinID:■ The position (number) of the sample pin in the UniPuck.
■ It is also possible to enter a group of pins (described later)■ If there is a duplicate PuckID and PinID, the "Confirmation required" line
displays a [Warning]. Please eliminate the duplication.

●SampleName:■ Datasets with the same ‘SampleName’ are automatically merged withKAMO (basically, merging is applied to multi mode)■ Merging is also applicable for a full data set from a single crystal(single/helical)◆ Data sets are first processed individually with KAMO
◆ KAMO merges datasets with the same ‘SampleName’.
◆ Please let us know if you need to merge multiple full datasets
obtained with single/helical modes◆ “KAMO_buttagiri processing” (described in detail later) also merges
multiple data sets with the same SampleName.
■ DO NOT use characters that cannot be used for directory names on
Linux. SampleName is used as the name of the directory when merging.
(e.g. Space, parenthese, etc.)◆ Please name them with alphanumeric characters or “_” and “-”.

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●Objective:■ Select the purpose of the data collection from the pull-down menu
■ According to this selection, the program sets experimental conditions forraster scan and data collection.
■ Please see further details in the table below. If you have any questions,please feel free to contact us.

●Modes (see also following figures):■ multi◆ Collect multiple datasets from multiple microcrystals mounted on oneloop.
◆ Typically, a 5-10º wedge is collected from each crystal.
◆ Each dataset is acquired from the angle where the largest surface ofa loop is the most visible against X-ray with ±2.5–5.0º.
◆ Collected datasets are merged into the final dataset with KAMO.

45

■

helical◆ Data collection with the rotation method while moving the X-ray
irradiation point from the left to the right edge of the crystal.
◆ This mode is suitable when the crystal is larger than the beam size.
◆ A full data set is collected from one crystal.
◆ Crystal size and shape are automatically recognized from the resultof the 2D raster scan. If the crystal size is less than twice the beamsize (e.g., 40 µm or less for 20-µm beam at BL45XU), the mode will
be automatically switched to single mode
(if you are not sure whether the crystal size is for single or helicalmodes, please choose helical)

6■●single■ Single-point irradiation (full data set from single crystal)■ We recommend that you do not select this option unless you would
like to increase data-collection speed, such as for proprietary
beamtime

HA:■ Select this option if the crystal contains heavy atoms and you need to use
anomalous scattering for phasing■ Select "no" if the crystal does not contain heavy atoms or you do not need
to use anomalous scattering for phasing■ On the sample sheet, when you select heavy atom from the pull-down
menu, the "Wavelength" field shows the wavelength that is about 0.001
Å shorter than the literature edge wavelength of the heavy atom (socalled 'peak wavelength'). This is used as a suggested value for SAD
measurement. However, we are not sure that the value is suitable foryour objective. Please input your preferred value in the column of'wavelength' if you like (see also ‘Wavelength’ section)■ If "no" is selected, 1.000 Å will be entered.

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●Wavelength:■ Wavelength for raster scan and data collection.
■ Please be informed that when HA is selected, it will be automaticallychanged to the wavelength near the peak one. If you input thewavelength manually here, the wavelength derived from HA selection willbe overridden.
■ Available wavelength range depends on beamlines; please refer the link
below. If you have further questions, please use the contact address.
http://bioxtal.spring8.or.jp/ja/BL/SPring8_PX_beamlines.pdf●Hor. scan length [µm]:(Please read the document below carefully with figures.)■ This parameter is used for defining the 2D raster scan area after loop
centering by INOCC.
◆ INOCC defines the 2D raster scan area after loop centering.
◆ This parameter defines the length of the raster scan area in thehorizontal direction (parallel to the rotation axis of the goniometer)◆ ZOO scans from the tip of the loop by this value (unit in µm)◆ It is recommended to enter the longitudinal length of the loop withsome margin.
◆ The length of the vertical direction is automatically determined by theshape of the loop with INOCC.

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●Resolution limit [Å]:■ The maximum resolution available based on the "Beamline" and"Wavelength" is displayed. Please override these values byreferring to the instructions below.
■ Resolution at the detector edge for data collection.
■ This parameter defines the camera length.
■ Enter the resolution of "this is more than enough".
■ Of course, you can also enter "3" if it is the first irradiation for the crystal
and is not expected to diffract to "3 Å".
■ If the longest cell parameter is quite long (e.g., >300 Å), you should
consider this value carefully. In this case, please let us know.
■ If not specified, we assume this value to be 2.0 Å.

●Beam size [um] (Order: HxV, horizontal x vertical):■ Reference
http://bioxtal.spring8.or.jp/ja/BL/SPring8_PX_beamlines.pdf■ Available beam sizes depend on each beamline; please refer to theabove. If you have further questions, please use the contact address.
■ For the beam size "HxV", set the center letter to lowercase "x". DO NOT
include “space” between characters.
◆ Example: 10x15

9●Crystal size [µm]:■ crystal size
■ This parameter is important in multi mode because it will be the shortest
distance between the exposure points◆ (e.g.) If one crystal is larger than this parameter, more than two
datasets will be collected from one crystal (the exposure point is ofcourse different) (see figure below).
◆ If the crystals are needle-shaped or columnar, please input theshortest length of the crystal shape.
◆ if it is set to the value less than "Beam size", the "Confirmation
required" line displays a [Warning].
■ In the case of helical/single mode data collection, crystal shape is
automatically determined from the results of the raster scan, so please
enter the approximate value for reference.

●# of crystals/loop:■ Number of crystals in the loop (rough estimation is okay)◆ In multi mode, this number defines the maximum number of datasets
per loop.
◆ In multi mode, if the value is set to “19 or less”, the "Confirmation
required" line displays a [Warning]. It is recommended to set thevalue to “20 or higher”.
◆ If you are not sure, please enter the possible number of crystals.
◆ If you are not sure AT ALL, you can use "50" or "100".
✧ Please keep in mind that the measurement time is affected bythis parameter.
10✧ (Note)
A catastrophic case with a large number:
There were a lot of substances that produced a strangediffraction pattern. It was incorrectly determined that there weremany crystals, and totally meaningless datasets were collected
for that particular number. In the end, much machine time wasconsumed, so it could not be completed data collection on time.
(Countermeasure)
It is better not to enter large values for crystals that are not wellcharacterized.
◆ In helical mode, if the value is set to “2 or higher”, the "Confirmation
required" line displays a [Attention]. The condition is only feasible if
two or more crystals do not enter the path of the X-rays.

◆ In single mode, if the value is set to “2 or higher”, the "Confirmation
required" line displays a [Warning]. The condition is not enforceable.
●Total osc. [deg.]:■ Oscillation range for data collection (deg./crystal).
■ In multi mode, the maximum value should be 15º/crystal (recommended10º/crystal). If the value is set to “16 or higher”, the "Confirmation
required" line displays a [Attention].
■ In helical mode, it is recommended to collect at least 360º (if the value isset to “359 or less”, the "Confirmation required" line displays a[Attention]). Currently, ZOO does not automatically evaluate the quality
or volume of the crystals. Thus, it is recommended to collect data with alarge total rotation angle. Therefore, it may be possible to obtain complete
data, even excluding the diffraction images from a bad region.
◆ In helical mode data collection, the irradiation point is translated.
Therefore, if there are crystal volumes with poor quality in the crystal(e.g. crystals are cracked, or stacked) , there is a risk of incomplete
data collection because we should throw away the data from such
crystal volumes after data processing.
11◆ If you collect a large amount of data, it may be possible to exclude
some of the data during data processing.

◆ For the data collected in helical mode, "data split processing" (known
as "buttagiri processing") can be applied in addition to normal dataprocessing.
◆ Data split processing involves selecting only the better data, as
described above.
◆ The data set is split into 30° (standard setting), processed as
independent data, then merged after clustering.
◆ You can compare the results with those from normal processing anddata split processing. Please choose the best results for structuralanalysis. Normal processing results are saved in the “_kamoproc/”
directory, and the data split processing results are saved in the“_kamo_30deg/” directory.
●Osc. Width:■ Oscillation width per frame (deg.) for data collection. Recommended
value is 0.1º

●Liquid N 2 :■ After the sample pin is mounted on the goniometer, wash off any frost on
the loop by using liquid N 2 . This can prevent or reduce the effect of theice ring. This takes about 15 sec. per sample.

Before dropping LN2

After dropping LN2
Acta Cryst. F76(12):616-622 (2020).
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●Pin Type:■ Select a sample pin type (SPINE, ALS+SSRL, Copper, or No-wait).
■ It has been determined that the sample pins extend to a max. of 50 μm
in about 5 min after being mounted on the goniometer. You can define
the waiting time for letting the pin extend (please refer the CAUTION
section at the end of this document)■ Recommendation: SPINE: 10 sec., ALS+SSRL: 20 sec., Copper: 60 sec,
No-wait: 0 sec.
■ If you would like to define a different waiting time, please use thecontact address.
The degree of elongation depends on the type of sample pin; theCopper type seems to be the worst. The SPINE type is recommended
because it is less elongated.

●Zoom Capture:■ In addition to the standard magnification, the crystal images are also
taken at high magnification, which takes about 5 sec.

<Pin ID setting method>1) Specify the range with a hyphen
1-16
2-5
14-162) In case of skipping
Continue the number with "." "+" ";" (Period, plus, semicolon).
(Prohibited character ",". Because it is a CSV file, insertion of comma destroys
the setting of sample sheet.)
Examples:1 + 5 + 10 + 15 (Measures 1 and 5, 10 and 15)
1.5.10.15 (measures 1, 5, 10, and 15)
1; 5; 10; 15 (measures 1, 5, 10, and 15)
1-5 + 10-15 (measures 1 to 5 and 10 to 15)
1-5; 10-15 (measures 1 to 5 and 10 to 15)
1-5.10-15 (measures 1 to 5 and 10 to 15)
As software, the character string of "column of pin ID" is first separated by a ".”"+" ";", followed by the range-specification character hyphen. The pin ID cannot
be set beyond the pack.
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<The following information is for those who want to know more detail>Setting the scanning conditions for difficult crystals→ under an assumption that they are not salt crystals
As of Jul 19, 2019
(Set the dose of raster scan to 1 MGy and the dose of data collection to 9
MGy, etc.)⚪ For example, if we use a 10 x 10 µm beam, let us say that Flux uses a9.7E12 phs/sec beamline.
⚪ Flux density is 9.7E12/10.0/10.0 = 9.7E10 photons/um2⚪ For a wavelength of 1 Å, 10 MGy is approximately equivalent to 2E10photons/um2⚪ This does is reached in 2E10/9.7E10 = 0.206 sec.
⚪ If you want to scan 1 MGy, this 1/10 exposure time is necessary.
■ Exposure time of 0.02 sec.
⚪ In this case, the scanning time is about a 400-µm loop.
■ 400 µm/10 µm step = 40 points/line■ 40 points * 0.02 sec/points = 0.8 sec/line■ The number of lines is 400 µm/10 µm = 40 lines with about 2 sec ofwrapping.
■ 40 lines * (2 + 0.8) sec/line = 112 sec■ The number of crystals found in this will take a few minutes of datacollection time.
■ 30 crystals * 10 sec = 300 sec■ Therefore, if there are 30 crystals in the 400-µm loop, therequired calculation is 112 sec + 300 sec = 412 sec/loop■ It is about 7 min/loop, so it is about 2 hrs per pack.

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<FAQ>

15

<CAUTION>
It is not recommended to use the sample pin with copper (shown in the figurebelow) for automated data collection.

Right)
A composite photo of the sample loop mounted on the pin with copper after 0 and
5 min.
The loop shifted 50 um horizontally and 36 um vertically within 5 min. Suchsignificant elongation occurs in a pin with copper. We are analyzing other typesof pins as well.

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