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Towards a compact and precise sample holder for macromolecular crystallography

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Most of the sample holders currently used in macromolecular crystallography offer limited storage density and poor initial crystal-positioning precision upon mounting on a goniometer. This has now become a limiting factor at high-throughput beamlines, where data collection can be performed in a matter of seconds. Furthermore, this lack of precision limits the potential benefits emerging from automated harvesting systems that could provide crystal-position information which would further enhance alignment at beamlines. This situation provided the motivation for the development of a compact and precise sample holder with corresponding pucks, handling tools and robotic transfer protocols. The development process included four main phases: design, prototype manufacture, testing with a robotic sample changer and validation under real conditions on a beamline. Two sample-holder designs are proposed: NewPin and miniSPINE. They share the same robot gripper and allow the storage of 36 sample holders in uni-puck footprint-style pucks, which represents 252 samples in a dry-shipping dewar commonly used in the field. The pucks are identified with human- and machine-readable codes, as well as with radio-frequency identification (RFID) tags. NewPin offers a crystal-repositioning precision of up to 10 µm but requires a specific goniometer socket. The storage density could reach 64 samples using a special puck designed for fully robotic handling. miniSPINE is less precise but uses a goniometer mount compatible with the current SPINE standard. miniSPINE is proposed for the first implementation of the new standard, since it is easier to integrate at beamlines. An upgraded version of the SPINE sample holder with a corresponding puck named SPINEplus is also proposed in order to offer a homogenous and interoperable system. The project involved several European synchrotrons and industrial companies in the fields of consumables and sample-changer robotics. Manual handling of miniSPINE was tested at different institutes using evaluation kits, and pilot beamlines are being equipped with compatible robotics for large-scale evaluation. A companion paper describes a new sample changer FlexED8 (Papp et al. , 2017, Acta Cryst. , D73, doi:10.1107/S2059798317013596).
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research papers
Acta Cryst. (2017). D73, 829–840 https://doi.org/10.1107/S2059798317013742 829
Received 18 April 2017
Accepted 25 September 2017
Edited by E. F. Garman, University of Oxford,
England
Keywords: sample holder; NewPin; miniSPINE;
SPINEplus; high density; high precision;
SmartMagnet.
Supporting information:this article has
supporting information at journals.iucr.org/d
Towards a compact and precise sample holder for
macromolecular crystallography
Gergely Papp,* Christopher Rossi, Robert Janocha, Clement Sorez, Marcos Lopez-
Marrero, Anthony Astruc, Andrew McCarthy, Hassan Belrhali, Matthew W. Bowler
and Florent Cipriani
European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble,
France. *Correspondence e-mail: gpapp@embl.fr
Most of the sample holders currently used in macromolecular crystallography
offer limited storage density and poor initial crystal-positioning precision upon
mounting on a goniometer. This has now become a limiting factor at high-
throughput beamlines, where data collection can be performed in a matter of
seconds. Furthermore, this lack of precision limits the potential benefits
emerging from automated harvesting systems that could provide crystal-position
information which would further enhance alignment at beamlines. This situation
provided the motivation for the development of a compact and precise sample
holder with corresponding pucks, handling tools and robotic transfer protocols.
The development process included four main phases: design, prototype
manufacture, testing with a robotic sample changer and validation under real
conditions on a beamline. Two sample-holder designs are proposed: NewPin and
miniSPINE. They share the same robot gripper and allow the storage of 36
sample holders in uni-puck footprint-style pucks, which represents 252 samples
in a dry-shipping dewar commonly used in the field. The pucks are identified
with human- and machine-readable codes, as well as with radio-frequency
identification (RFID) tags. NewPin offers a crystal-repositioning precision of up
to 10 mm but requires a specific goniometer socket. The storage density could
reach 64 samples using a special puck designed for fully robotic handling.
miniSPINE is less precise but uses a goniometer mount compatible with the
current SPINE standard. miniSPINE is proposed for the first implementation of
the new standard, since it is easier to integrate at beamlines. An upgraded
version of the SPINE sample holder with a corresponding puck named
SPINEplus is also proposed in order to offer a homogenous and interoperable
system. The project involved several European synchrotrons and industrial
companies in the fields of consumables and sample-changer robotics. Manual
handling of miniSPINE was tested at different institutes using evaluation kits,
and pilot beamlines are being equipped with compatible robotics for large-scale
evaluation. A companion paper describes a new sample changer FlexED8 (Papp
et al., 2017, Acta Cryst.,D73, 841–851).
1. Introduction
With the emergence of cryocrystallography (Teng, 1990) as a
standard technique in macromolecular crystallography (MX),
various sample holders for protein crystals were developed or
adapted from existing supports for crystallographic cryogenic
data collection (Garman & Owen, 2006). The ‘top-hat’ design,
exemplified by the Hampton Research Magnetic Base, has
proved to be highly successful and over many years has
evolved into several similar designs that were subsequently
standardized for the needs of robotic sample mounting
(Cohen et al., 2002; Karain et al., 2002; Snell et al., 2004;
Cipriani et al., 2006). Among them, the European SPINE
standard was established in 2005 as an evolution of existing
commercial cap-and-vial models. This standard played a key
ISSN 2059-7983
role in beamline automation in Europe and made it possible to
collect data at different European beamlines with minimal
compatibility problems (Beteva et al., 2006). Nevertheless, as
with all existing sample-holder standards, the SPINE standard
has become a limiting factor at high-throughput beamlines.
At the most recent third-generation synchrotron MX
beamlines, the time needed to centre and align a crystal with
the X-ray beam is comparable to the time needed to collect an
X-ray data set (Svensson et al., 2015; Casanas et al., 2016). This
significantly impairs beamline efficiency. The situation will
become worse at future fourth-generation light sources where
only tens of milliseconds will be necessary for a typical MX
data collection. This overhead could be significantly reduced
by using sample holders that provide precise initial crystal
positioning, in particular for crystals harvested by automated
systems that can record a crystal position in the sample holder
(Cipriani et al., 2012; Deller & Rupp, 2014; Zander et al., 2016).
Improved initial crystal positioning will reduce crystal-
alignment time for both optical (Lavault et al., 2006; Pothineni
et al., 2006) and X-ray-based methods (Svensson et al., 2015;
Song et al., 2007; Bowler et al., 2016). Similarly, for serial data
collection from microcrystals, the region of interest can be
directly scanned after the sample holder has been mounted on
the goniometer and the recorded alignment offset has been
applied (Zander et al., 2015; Gati et al., 2014). An additional
limiting factor is the size of the sample holders, as this directly
impacts sample-storage density and the associated storage and
transport costs. In Europe, most sample changers are based on
six-axis industrial robotics and use SPINE or uni-puck (http://
smb.slac.stanford.edu/robosync/Universal_Puck/) containers.
Maximum storage density is currently obtained with the uni-
puck (16 samples), allowing up to 112 samples (in seven
uni-pucks) to be sent in a single CX100 shipping dewar.
Modern high-throughput beamlines now use sample changers
equipped with dewars that can hold up to 384 samples (24 uni-
pucks; Bowler et al., 2015; Nurizzo et al., 2016; Owen et al.,
2016; Russi et al., 2016). This capacity is obtained at the cost of
using large sample-changer dewars. Storage density can also
be increased by using specific containers such as the SSRL
cassette used by the SAM sample changer (Cohen et al., 2002;
Russi et al., 2016) that allow up to 192 Hampton Research
CrystalCap-like sample holders to be placed in a CX100
shipping dewar. Nevertheless, SAM pucks have not been
considered in Europe, probably because access from the side
of the container is too different from the widespread top
access and would require the adaptation of current robotic
sample changers. Similarly, a proprietary high-density sample-
holder system, which is used at the SPring-8 synchrotron, has
been developed (Ueno et al., 2004). The SPACE system is
based on high-density plastic pins with two screw threads for
transport and mounting on the goniometer. However,
extending its usage to Europe would require the use of specific
shipping dewars and a major integration effort at beamlines.
In common with other existing sample-holder standards, the
European SPINE standard has two fundamental limitations.
Firstly, the size of the sample holder limits the sample-storage
density to 112 samples per transport dewar. Secondly, the
mechanical tolerances and absence of an orientation index
induce a variation of up to 1 mm in the initial crystal posi-
tioning at the sample position upon mounting. This also limits
the repositioning precision upon loading/unloading, which
makes loop and/or crystal alignment necessary before each
data collection (Lavault et al., 2006; Pothineni et al., 2006;
Svensson et al., 2015). Furthermore, the cost of the sample
holder can limit the number of samples prepared and there-
fore the usage of high-throughput techniques for synchrotron
data collection (Abola et al., 2000). Reusable sample-holder
bases can contribute to cost reduction but require additional
manpower to clean and recycle the sample holders. Conse-
quently, in 2009 a feasibility study for a compact, precise and,
as far as possible, cost-effective sample holder with corre-
sponding manual and robotic handling tools compatible with
six-axis industrial robots was initiated at the EMBL Grenoble
Outstation. This project has been supported since 2011 by the
BioStruct-X FP7 European programme, with the aim of
defining a new European sample-holder standard. The kick-
off meeting was held in December 2011 in Hamburg with the
participation of seven partners: (i) SLS (Paul Scherrer Insti-
tute, Villigen, Switzerland); (ii) BESSY II (Helmholtz-
Zentrum Berlin fu
¨r Materialien und Energie, Berlin,
Germany); (iii) MAX-IV laboratory (Lund, Sweden); (iv)
EMBL@PETRA-III (European Molecular Biology Labora-
tory, Hamburg, Germany); (v) ESRF (The European
Synchrotron Radiation Facility, Grenoble, France); (vi) DLS
(Diamond Light Source Limited, Oxford, England) and (vii)
EMBL Grenoble (European Molecular Biology Laboratory,
Grenoble, France). The discussions conducted after the initial
development stages revealed the importance of beamline-
integration aspects and led to the definition of two new
sample-holder designs, NewPin and miniSPINE, together with
an improved version of the SPINE standard called SPINEplus.
All of the corresponding containers are compatible with a
specific sample-changer dewar slot that can also receive
standard uni-pucks. Migration from the SPINE standard to
NewPin, the ultimate sample-holder version proposed, could
then be facilitated. All of the sample-holder models have been
tested on a beamline under real conditions, using a sample
changer built around a storage dewar (Papp et al., 2017), a
six-axis Sta
¨ubli TX60L industrial robot and corresponding
grippers. The miniSPINE model was selected for large-scale
testing as it is easier to integrate with existing beamline
robotics and can be handled manually. 12 evaluation kits were
manufactured and distributed to the project partners, other
interested synchrotrons and institutes (IBS, NSLSII, Photon
Factory and SPring-8) and to industrial companies working in
the field. The feedback obtained concerning the ergonomics of
the manual tools has been integrated into the final design of
the miniSPINE version. Here, we describe the design, testing
and results from the use of the three sample holders proposed.
2. Experimental details
The first phase of the development process focused on the
NewPin sample holder, which is a simple pin of 22 mm in
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830 Papp et al. A compact and precise sample holder Acta Cryst. (2017). D73, 829–840
length and 1.9 mm in diameter. Its design fully meets the
requirements for high storage density (36 samples per puck
and up to 64 for the model anticipated for fully automated
robotic pin handling) with a repositioning precision of 10 mm
(Papp et al., 2017). Nevertheless, after the initial evaluation it
became clear that an intermediate version that was easier to
handle manually and to integrate at beamlines would be
required. Therefore, the miniSPINE sample holder was
developed: a compact version of the SPINE sample holder
that provides high storage density (36 samples per puck) and is
easier to integrate on existing beamlines. The type of crystal
mount (i.e. the loop or support that will hold the crystal) of
the sample holders is not defined as they can receive any
commercially available nylon loop/LithoLoop (Molecular
Dimensions, Suffolk, England) or MicroMounts (MiTeGen,
Ithaca, USA) or can be customized for specific requirements,
such as for the CrystalDirect harvester (Cipriani et al., 2012;
Zander et al., 2016). Attempts were made to design a vial for
both new sample-holder types. Different vial-to-pin coupling
methods were explored but insoluble handling problems, as
well as the difficulties anticipated in manufacturing the vials at
an affordable cost, led this option to be abandoned. Conse-
quently, a closed robot gripper that acts as a cold buffer was
developed to keep the crystals below 100 K during transfers in
ambient air and to protect them from ice contamination. This
gripper is compatible with both the NewPin and miniSPINE
sample holders. Corresponding storage pucks and manual
handling tools were also developed for the NewPin and
miniSPINE sample holders. Both pucks can store 36 sample
holders, leading to an increase in sample density in the widely
used CX100 dry-shipping dewars by a factor of five versus the
SC3-SPINE pucks (Cipriani et al., 2006) and of more than two
versus uni-pucks. The uni-puck footprint standard (http://
smb.slac.stanford.edu/robosync/Universal_Puck) was adopted
to ensure backwards compatibility with uni-pucks and to
facilitate the migration of sample changers already installed at
European MX beamlines, such as CATS
(Jacquamet et al. 2009), G-Rob (Ferrer
et al., 2013), BART (Diamond Light
Source, England) and ACTOR Rigaku
systems (http://smb.slac.stanford.edu/
robosync/). A specific dewar slot that is
compatible with uni-pucks and that
ensures precise positioning of the
NewPin and miniSPINE pucks has also
been developed. Furthermore, to offer
an interoperable line of sample holders
and pucks, we decided to slightly modify
the SPINE standard, creating the
SPINEplus sample holder and a corre-
sponding sample-storage puck. The
three new supports and associated
pucks are shown in Fig. 1. The three
sample-holder models are designed to
be pre-oriented in specific storage racks
to be further manipulated with known
orientation (see x2.4).
Finally, it is possible to have an automated beamline
environment that is compatible with both miniSPINE and
SPINE sample holders, thus enabling a smooth transition
between the actual SPINE standard, miniSPINE and subse-
quently NewPin. The following sections describe the new
sample holders, their storage pucks and handling tools.
2.1. SPINEplus
SPINEplus (Supplementary Fig. S1) has been designed to
facilitate migration from the current SPINE standard to
miniSPINE and from there to NewPin. The SPINEplus sample
holder is backwards-compatible with the popular SPINE
sample-holder standard. Its corresponding puck has a uni-
puck footprint and is machine-identifiable. This is a key
feature when different sample-holder models are used on the
same beamline. The technical specifications are available in
the Supporting Information.
2.1.1. The SPINEplus sample holder. Compared with the
SPINE sample holder, SPINEplus (Fig. 2a) includes the
following new features: (i) an orientation slot at the base of
the cap for orientation indexing on goniometers, in pucks or
on automated harvesting systems; (ii) a tighter tolerance of the
inner diameter of the cap for compatibility with new robotic
tools and (iii) flats and holes on each side of the cap to grab,
flip and store the sample holder in a SPINEplus puck after
crystal harvesting using a manual harvesting tool (Fig. 2b).
Despite these modifications, the SPINEplus cap remains
compatible with the SPINE vials and pucks, as well as with
uni-pucks.
2.1.2. The SPINEplus puck. The SPINEplus puck (Fig. 2c)
has a capacity of 16 samples, permitting the storage of up to
112 sample holders in one CX100 dry-shipping dewar.
It is composed of (i) a ferromagnetic stainless-steel base
containing 16 magnets on the upper side to hold the pins, and
four magnets on the bottom side to maintain the puck in a
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Figure 1
Overview of the presented sample holders with main dimensions and their corresponding 36-well
pucks: (a) SPINEplus, (b) miniSPINE and (c) NewPin shown with a supplementary 64-well puck
version.
ferromagnetic dewar slot; (ii) an aluminium body with 16 wells
to keep the sample in liquid nitrogen during transport, even
when inclined; (iii) an identification pod to facilitate puck
tracking and (iv) a nonferromagnetic stainless-steel cover to
protect crystals from ice contamination during transport. The
pins are stored vertically with crystals at the top, locked by the
magnets. The pins can be inserted into the puck wells
(regardless of their angular orientation) using a specific
manual harvesting tool (Fig. 2b) or the gripper of a robotized
system, such as a sample changer or the cryostorage robotics
of an automated crystal harvester (Papp et al., 2017). The
identification pod described in x2.5
combines human-readable and Data-
matrix codes, and can receive an RFID
tag. The SPINEplus puck fits into
specific dewar slots that can also receive
uni-puck, miniSPINE and NewPin
pucks (Papp et al., 2017).
2.1.3. The SPINEplus handling tools.
Two manual tools have been developed
to manipulate the SPINEplus sample
holders: the crystal-harvesting tool (Fig.
2b) to hold the pins during crystal
harvesting and to store them in pucks,
and the pin-extracting tool (Fig. 2d)to
remove the pins from the pucks. Both
tools are used in a similar manner to the
handling schemes adopted for NewPin
and miniSPINE described in x2.2.4,
except that no pin-grabbing or puck-
loading assistants are necessary.
2.2. miniSPINE
The miniSPINE design (Supplemen-
tary Fig. S3) enables the storage of up to
36 samples in a puck and can offer initial
crystal positioning and positioning
repeatability within 100 mm, depending
on the transfer robotics used. The
miniSPINE sample holder is a compact,
SPINE-type sample holder with a 7 mm
diameter ferromagnetic base (Figs. 1b
and 3a). Similar to the SPINE model, it
is held on a goniometer with a magnetic
mount. Both SPINE and miniSPINE
can be used consecutively on a beamline
using a special goniometer mount
(Fig. 4aand Supplementary Fig. S6).
miniSPINE has been designed for high
storage density and has the advantage
of easier integration compared with
NewPin at beamlines equipped with
goniometers that use magnets to hold
the sample, in particular on those
compatible with the SPINE sample-
holder standard. Unlike NewPin, the
positioning precision of the miniSPINE sample holder on a
goniometer is highly dependent on the precision of the
transfer robotics (Papp et al., 2017). The technical specifica-
tions of the pins and puck are available in the Supporting
Information.
2.2.1. The miniSPINE sample holder. Two designs for the
miniSPINE sample holder, miniSPINE (MS) and mini-
SPINErf (MSrf) (Figs. 3aand 3b), are proposed. Mechanically
compatible, they offer identification using either a Datamatrix
label or an RFID tag. MS is a single piece of ferromagnetic
metal that can receive different commercially available crystal
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832 Papp et al. A compact and precise sample holder Acta Cryst. (2017). D73, 829–840
Figure 2
SPINEplus. (a) Sample holder with orientation notch, flats and holes for compatibility with the
manual harvesting tool. (b) Harvesting tool. (c) Puck with identification pod, 16 individual pin wells,
puck- and pin-holding magnets, cover and orientation groove. (d) Pin-extracting tool. Manual
harvesting steps are indicated in italics.
Figure 3
miniSPINE sample holder and goniometer mount. (a) Three-dimensional view of miniSPINE
showing the optional Datamatrix, pin-grabbing area, orientation flat and optional holes for the
manual harvesting tool. (b) Three-dimensional view of miniSPINErf shown with an RFID tag. (c)
SmartMagnetP goniometer mount shown with a miniSPINE sample holder.
mounts set on standard 0.63 mm needles, such as nylon loops,
MicroMounts or LithoLoops. It can optionally be identified
with a 14-point ECC200 Datamatrix code engraved on its base
for tracking purposes. MSrf is composed of two parts: a base
that is similar to the base of MS (shown in purple in Fig. 3a),
and a tube (Fig. 3b, salmon) that is similar to the NewPin
sample holder (x2.3). The MSrf model can host an RFID tag
and directly receive crystal mounts on its 0.63 mm tip. The
distance from the base of the miniSPINE sample holder to the
crystal is fixed at 19.8 mm. This ensures compatibility with
goniometer mounts designed to receive both SPINE and
miniSPINE sample holders (x2.6). The optional holes on each
side of the pin-support base (Figs. 3aand 3b) allow handling
with a manual harvesting tool (Fig. 4b) and the ability to flip
between harvesting and storing positions (Fig. 4). Further-
more, when stored in a supply rack, the angular orientation of
the miniSPINE sample holders can be fixed using the orien-
tation flat (Fig. 3a). This feature is essential when automated
harvesting methods that require a fixed pin orientation are
used (Cipriani et al., 2012). A 0.2 mm shoulder at the base of
the sample holder (Fig. 3a) reduces the surface area of contact
with the support (the bottom of the well in the puck or the
goniometer) to a ring. This minimizes the effect of ice or
particle contamination, thus improving the positioning preci-
sion and stability of the pins on goniometers and in pucks. The
integrity of crystals during shipping and robot handling is
ensured by individual wells in the pucks (Fig. 4e) and by the
closed robot grippers that act as cryo-tongs during sample
transfer (Papp et al., 2017). The robot grips miniSPINE
supports by the 1.9 mm diameter section; therefore, the same
robot gripper can be used to handle both the NewPin and
miniSPINE sample holders (Papp et al., 2017).
2.2.2. The miniSPINE goniometer mount. The miniSPINE
sample holder, like the SPINE sample holder, is held
magnetically on the goniometer. Although a simple perma-
nent magnet is sufficient to hold a pin on a goniometer, to
ensure reliable sample transfer, beamlines are usually
equipped with electromagnets that both hold and detect
sample holders, so-called SmartMagnets (Cipriani et al., 2006).
These devices were developed for the SPINE sample holder
and have a concentric magnetic pole topology that is not
compatible with miniSPINE. In this original SmartMagnet, the
magnetic poles are coaxial. The first pole is in the centre and
the second is a ring situated where the SPINE sample holders
sit. When mounted, a SPINE cap closes the magnetic circuit,
giving a sufficient holding force. However, this topology does
not provide sufficient force to hold miniSPINE as it is only in
contact with the central pole. Therefore, we have developed a
new type of SmartMagnet with parallel magnetic poles, the
SmartMagnetP (Supplementary Fig. S6), that is compatible
with both SPINE and miniSPINE. As shown in Supplemen-
tary Fig. S6, the topology of the SmartMagnetP is such that the
two poles of the electromagnet are parallel. In this config-
uration the magnetic circuit is closed for both SPINE and
miniSPINE, providing sufficient holding force for both sample
holders. The SmartMagnetP is geometrically and electrically
interchangeable with the classical SmartMagnet, making a
goniometer compatible with both SPINE and miniSPINE
sample holders. The SPINE sample holder is guided and
approximately centred by the central part of the SmartMagnet
when mounted on the goniometer mount (x2.6), whereas the
miniSPINE sample holder sits on the flat end of the Smart-
MagnetP (Fig. 3c). Therefore, no mechanical centring is
applied and the positioning precision of the miniSPINE pin
primarily depends on the precision of
the handling robotics. When manual
mounting on a goniometer is necessary,
for example when harvesting crystals at
a beamline, an adaptor ring (not shown)
can be mounted on the SmartMagnetP
to facilitate mounting of the sample
holder at the centre of the goniometer
mount. Specific goniometer mounts
with positioning stops could also be
developed to improve the initial and
repositioning precision of miniSPINE
when compatibility with SPINE or
SPINEplus sample holders is no longer
necessary (Papp et al., 2017).
2.2.3. The miniSPINE sample-storage
puck. The miniSPINE puck (Fig. 4e)
can hold up to 36 miniSPINE sample
holders in a format compatible with dry-
shipping dewars. It is composed of (i) a
ferromagnetic stainless-steel base with
36 hollow magnets to maintain the pins
in position and to hold the puck in place
when it is installed on a ferromagnetic
dewar slot; (ii) an aluminium body with
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Figure 4
miniSPINE puck and manual handling tools. (a) Pin-grabbing assistant. (b) Manual harvesting tool.
(c) Puck-loading assistant. (d) Pin-extracting tool (also compatible with the NewPin sample holder).
(e) Puck with identification pod, 36 individual pin wells, puck- and pin-holding hollow magnets,
cover and orientation groove. Manual harvesting steps are indicated in italics.
36 wells that maintain the samples in liquid nitrogen during
transfer of the puck between dewars; (iii) an identification pod
to facilitate puck tracking and (iv) a nonferromagnetic stain-
less-steel cover to minimize ice contamination during trans-
port in air. The pins are stored vertically with the crystals
pointing up. A taper in the diameter at the bottom of each well
enables precise positioning of the miniSPINE pins inside the
puck upon insertion. The identification pod combines human-
readable and Datamatrix codes, and can receive an RFID tag
(Fig. 4e). The miniSPINE puck is compatible with specific
dewar slots that can also receive uni-puck, SPINEplus and
NewPin pucks (Papp et al., 2017). Up to 288 pins can be
transported in a CX100 dewar using the eight-puck top-access
canister or up to 252 using the seven-puck shelved canister
(x2.7).
2.2.4. The miniSPINE handling tools. A set of manual tools
have been developed to handle miniSPINE pins (Fig. 4 and
Supplementary Fig. S5). In April 2015 miniSPINE evaluation
kits were distributed to partner sites and industrial partners in
order to assess the ergonomics of manual handling. Requests
and suggestions from project partners were addressed and
integrated to form the design presented here.
As miniSPINE is considerably smaller than the current
mounts, a puck-loading assistant was developed (Fig. 4c)to
facilitate loading the pins into the puck during manual crystal
harvesting (Supplementary Video S1). It is composed of a
support with a reflective base installed in a dewar (26 B/BE,
KGW Isotherm, Karlsruhe, Germany) and a light source
equipped with an optical fibreglass light guide with a 7.8 mm
diameter tip. The puck is placed in the support and the tip of
the fibre is inserted in the centre of the puck. The dewar can be
filled with liquid nitrogen before or after the loading assistant
and miniSPINE puck are installed. The light emitted by the
fibre is diffused by the bottom of the assistant and passes
through the hollow magnets of the wells, clearly identifying
wells that are free or occupied. Crystal harvesting (Fig. 4)
starts by inserting an empty pin in the grabbing assistant (Step
1). Held by a magnet and properly oriented, the pin is placed
in the harvesting tool and further rotated until it locks at 45in
a groove, the position used to harvest crystals from crystal-
lization trays (Step 2). After harvesting and cryoprotection,
the pin can be plunged into liquid nitrogen (Step 3) and then
flipped using the pin-flipping slot of the puck-loading assistant
located on the side of the puck slot (Step 4) below the liquid-
nitrogen level, allowing the pin orientation to be changed to
be placed into the puck. When aligned with the harvesting
tool, the pin with crystal can be inserted into an empty puck
slot (Step 5). It is important to vitrify the crystal before flip-
ping. This ensures faster cooling and secures the attachment of
the crystal in the cryo-mount. The level of liquid nitrogen in
the dewar should be kept at a minimum of 5 cm above the
flipping slot to ensure safe crystal handling. After processing,
the pins can be removed from the puck using the pin-
extracting tool (one pin at a time). The holes on the bottom of
the pucks can also be used to simultaneously push all of the
pins out of the pucks. The design of a pin-extracting tool with
36 fingers is shown in Supplementary Fig. S12. It can be used
without the handle (fingers pointing up) with the puck
installed above to safely recover each individual pin with
tweezers, or with the handle to clear the puck.
2.3. NewPin
NewPin (Fig. 5aand Supplementary Fig. S7) is our ultimate
sample-holder proposition. The sample holder is a single pin
on which the crystal mount is directly attached. Specifically
adapted for fully automated harvesting and data-collection
pipelines, it should fit perfectly into future entirely robotic
systems that cover crystal harvesting and data collection. In
this case, the storage density could reach up to 64 sample
holders in a puck and up to 512 samples in the eight-puck top-
access canister compatible with the CX100 shipping dewar.
Manual crystal harvesting is difficult but possible using a set of
handling tools and pucks with a capacity reduced to 36 sample
holders currently proposed here. A possible design for a 64-
sample version is proposed in Fig. 6(e). The NewPin sample
holder fits in a specific goniometer mount containing a socket
(Figs. 5band 5c; Supplementary Fig. S8) that allows a crystal
positioning repeatability of better than 10 mm. The technical
specifications are available in the Supporting Information.
2.3.1. The NewPin sample holder. NewPin (Fig. 5a) consists
of a single needle of 22 mm in length and 1.9 mm in diameter,
reducing to 0.64 mm diameter at one end to receive a cryo-
loop or equivalent crystal support. At the other end of the
needle, or pin base, a 4 mm flat plane is provided to fix the
angular orientation on a goniometer mount, inside a puck and,
potentially, on a crystal harvester mount. The bottom of the
pin base is flat to fix its axial position. A specific auto-aligning
and auto-orienting mechanical goniometer mount (x2.3.2) has
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834 Papp et al. A compact and precise sample holder Acta Cryst. (2017). D73, 829–840
Figure 5
NewPin sample holder and goniometer mount. (a) Three-dimensional
view of the NewPin sample holder with the orientation flat, two optional
grabbing holes for the manual harvesting tool, the pin-grabbing area, an
optional RFID tag and the 0.635 mm tip that can receive different crystal
mounts. (b,c) Principle drawing of the goniometer mount that shows the
compliant stop, the V-shaped guide and the orientation/locking pusher
been developed to reach a repositioning precision within
10 mm (Papp et al., 2017). Two holes on the sides are provided
to grab and flip the pin with a specific tool used for manual
harvesting (x2.3.4). Optionally, the pin can be hollow to
receive an RFID tag and facilitate sample tracking (x2.5). The
grabbing area for the robot is situated on the 1.9 mm diameter
part of the pin (Fig. 5a). For the reasons previously explained,
there is no vial associated with the NewPin sample holder. The
length of the pin was chosen to facilitate integration and
compatibility with existing goniometer mounts, and kappa
goniometers, compatible with SPINE sample holders (x2.6).
NewPin can be fabricated by machining, tube swaging or
stamping. The last method is the most cost-effective for large
production batches but requires a significant investment in
machine tooling.
2.3.2. The NewPin goniometer mount. The NewPin
goniometer mount (Figs. 5band 5c) is a mechanical socket that
automatically fixes the three-dimensional position of the pin
upon insertion, with automatic correction of the initial posi-
tioning and orientation. The auto-aligning system tolerates an
initial radial orientation error of up to 5. The socket
consists of a V-shaped guide, an orienting/locking pusher and a
compliant stop. In the prototype (Supplementary Fig S8), the
compliant stop and orientation finger are each composed of a
spring and a jack. For optimal three-dimensional positioning,
the pins should be inserted pre-oriented within 5and about
0.5 mm further in than the nominal position. This ensures that
upon release the compliant stop brings the pin back to the
nominal axial position, while the orientating/locking pusher
pushes it towards the V-shaped guide to ensure the correct
radial and angular position. A crystal-repositioning precision
better than 10 mm was obtained upon successive loading/
unloading of the same pin (Papp et al., 2017).
2.3.3. The NewPin puck. The NewPin puck (Fig. 6c) can
hold up to 36 NewPin sample holders in a format compatible
with dry-shipping dewars. It is composed of (i) a ferromagnetic
stainless-steel base containing 36 mechanical sockets to
maintain the pins in position and four magnets to hold the
puck in a ferromagnetic uni-puck dewar slot; (ii) an aluminium
body with 36 wells to maintain the samples in liquid nitrogen
during transport; (iii) an identification pod to facilitate puck
tracking and (iv) a nonferromagnetic stainless-steel cover to
minimize ice contamination during transport. The pins are
stored vertically with crystals pointing up and locked in the
sockets by locking/orienting springs (Fig. 6c). The pins are
plugged pre-oriented using a specific manual harvesting tool
or the gripper of a robotized system, for example a sample
changer or the cryostorage robotics of an automated crystal
harvester. The identification pod combines human-readable
and Datamatrix codes and can receive an RFID tag (x2.5). The
NewPin puck is compatible with specific dewar slots that can
also receive uni-puck, SPINEplus and miniSPINE pucks
(Papp et al., 2017). Up to 288 NewPins can be transported in a
CX100 dewar using the eight-puck top-access canister or up to
252 using the seven-puck shelved canister (x2.7). A prototype
with 66 pin slots was designed and the corresponding base was
manufactured (Supplementary Fig. S9). From this prototype, a
64-slot beta version was designed and specified (not manu-
factured) to illustrate the ultimate capabilities of NewPin
using test robotics. Nevertheless, it should be highlighted that
the absence of individual sample wells
keeps the crystals under liquid nitrogen
only within a small puck-inclination
range, thus making manual handling of
the pucks delicate. This effect is never-
theless mitigated by the presence of the
cover.
2.3.4. NewPin manual handling
tools. Three manual handling tools
have been developed: a pin-grabbing
support (Fig. 6a) that keeps the pin
firmly and correctly oriented to facil-
itate mounting on the harvesting tool, a
harvesting tool (Fig. 6b) to hold a pin
correctly during crystal harvesting, and
a pin-extracting tool (Fig. 6d) to remove
a pin from a puck. The harvesting tool is
a tweezer with a stud on each jaw that
fits into the two holes on each sides of a
pin. The harvesting process is similar to
the process described above for mini-
SPINE and is as follows: Step 1, the pin
is inserted into the pin-grabbing
support, gripped with the harvesting
tool and is oriented manually at 45to
facilitate harvesting; Step 2, the crystal
is harvested and cryoprotected; Step 3,
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Acta Cryst. (2017). D73, 829–840 Papp et al. A compact and precise sample holder 835
Figure 6
NewPin puck and manual handling tools. (a) Pin-grabbing support. (b) Harvesting tool. (c) Puck
with identification pod, 36 individual pin wells, puck-holding magnets, pin-orienting/holding springs,
cover and orientation groove. (d) Extracting tool (also compatible with miniSPINE sample
holders). Manual harvesting steps are indicated in italics.
the pin with its crystal is plunged into liquid nitrogen; Step 4,
the pin is flipped by 135, possibly using a flipping assistant
similar to the miniSPINE puck-loading assistant (Fig. 4c); Step
5, the pin with its crystal is inserted in a puck slot and released
by pressing the button situated on the handle of the tool. To
facilitate handling, the pins are locked in the harvesting and
storing positions. As with miniSPINE, it is important to vitrify
the crystal before flipping. This ensures faster cooling and
secures the attachment of the crystal in the cryo-mount.
Although manual harvesting is possible, NewPin is better
adapted to fully robotic handling, in particular when consid-
ering the difficulties of visualizing puck slots in liquid nitrogen.
2.4. Pin orientation
The precise orientation of the pins is particularly important
with crystal mounts that set the crystal off the central pin axis,
such as MicroMounts (MiTeGen) or in automated processes
where the initial sample-holder orientation needs to be fixed,
such as CrystalDirect (Zander et al., 2016). Therefore, we have
designed dedicated pin racks for each proposed sample-holder
model (Supplementary Fig. S13) where the pins are pre-
oriented. The specific pin slots for each sample-holder type
include orienting locks for NewPin, holes with orientation flats
for miniSPINE and orientation ridges for SPINEplus. It is also
possible to fix the orientation of the SPINEplus pins upon
manual mounting on a goniometer mount equipped with an
orientation finger. It should be noted that pin orientation is
intrinsic to the design of NewPin and must be respected upon
handling.
2.5. Sample identification
To facilitate sample tracking, different models of commer-
cial sample holders and pucks have been proposed with
optical identification tags such as barcodes, Datamatrix codes
or coloured rings. The reliability of such optical identification
methods often suffers from icing, surface degradation or fog,
and is sensitive to lighting conditions. To overcome these
issues, Rigaku Corporation (Tokyo, Japan) proposed and
produced sample holders with custom RFID-tag identifiers.
Nevertheless, their use remained limited and commercializa-
tion was stopped. Here, we propose to identify NewPin and
miniSPINE sample holders with low-frequency RFID tags,
possibly combined with Datamatrix codes. To identify pucks,
we propose pods that can include a human readable code, a
Datamatrix code and a high-frequency RFID tag.
The NewPin and miniSPINE radio-frequency (MSrf)
sample holders (Figs. 3band 5a) have been designed to receive
RFID tags of 1.4 mm in diameter and 8 mm in length. A low-
frequency glass tag was selected for reading though the body
of the pins (in stainless steel), as well as when the pins are in
sample-changer grippers. Standard tags from different manu-
facturers were tested. All showed a 30% frequency drift
between room and liquid-nitrogen temperatures. To make
them readable at both temperatures, the antennae of the tags
were modified for a 15% frequency detuning against the
nominal frequency at room temperature. This frequency
adjustment allowed the use of a unique standard RFID reader
antenna (tuned to 125 kHz) for both room and cryo-
temperature reading, simplifying future RFID reading
stations. The major remaining problem with the RFID glass
tags is a significant failure rate against temperature cycling
between room and liquid-nitrogen temperatures. A partner-
ship was therefore established with the company HID Global
IDT (Granges-Veveyse, Switzerland) with the aim of reducing
this failure rate to 0.1% after 100 cycles.
An identification pod for the NewPin puck (Fig. 6c),
miniSPINE puck (Fig. 4e) and SPINEplus puck (Fig. 2c)has
been defined. The pod combines a human-readable ID and a
Datamatrix label and can include a high-frequency RFID tag.
Preliminary temperature-cycling tests on one batch of 50
commercially available high-frequency RFID tags showed a
2% failure rate over 100 cycles. Further improvements of the
chips are required to reach a failure rate of 0.1% after 1000
cycles.
An important aspect of sample identification is the benefit-
to-cost ratio. The total manufacturing cost of a sample holder
is composed of three parts: the mechanical support, the crystal
mount and the identifier. Unlike the widely used Datamatrix
identification, the RFID tag-based identification proposed
here represents a significant part of the total fabrication cost
of a sample holder. On the other hand, the identification of a
puck is much more affordable as it represents only a few
percent of its total cost. Tracking projects with large numbers
of equivalent crystals does not necessarily require the identi-
fication of each sample. Puck identification is most often
sufficient. Similarly, fully automated environments can rely on
the position of the sample holders in the pucks (Bowler et al.,
2015). Conversely, ligand-screening applications, where each
crystal contains a different molecule, could benefit from
sample-holder identification.
2.6. Pin length
Pin length is the usual way to name the length of the sample
holder. At beamlines, it defines the distance from the gonio-
meter mount to the X-ray beam. The SPINE sample-holder
standard has a fixed pin length of 22 mm (from the base of the
cap to the crystal or beam position). Adopting a unique pin
length significantly reduced the beamline-compatibility issues
and facilitated the use of kappa goniometers presenting
limited tolerance against pin-length variations (Brockhauser et
al., 2013). Particular care was taken in the choice of the
NewPin and miniSPINE sample-holder lengths in order to
facilitate the design of compatible goniometer mounts (Fig. 7).
2.7. Puck-handling tools and dewar canisters
A common tool (Fig. 8a, Supplementary Fig. S10) was
developed to manipulate the NewPin, miniSPINE and
SPINEplus pucks. Three spring-loaded balls at the tip of the
tool grab the puck from a groove when the tool is inserted into
the centre of the puck. The puck can be released with or
without the cover by pressing the button at the top of the tool.
When the puck is held in the tool, a spring pushes a disc
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836 Papp et al. A compact and precise sample holder Acta Cryst. (2017). D73, 829–840
against the top side of the puck. The friction between this disc
and the puck allows the puck to be aligned with an orientation
finger in a sample-changer dewar slot. Additionally, two
canister designs are proposed for transport dewars. In the first
design, the eight-puck top-access canister (Fig. 8b), the pucks
are simply stacked and are directly accessible using a tool
similar to the puck-handling tool described above, but with an
extended handle. In this case, the pucks are accessible in first-
in-last-out (FILO) order. The second model, the seven-puck
shelved canister (Fig. 8c, Supplementary Fig. S10) provides
random puck access but requires an additional tool to handle
the pucks. Orientation fingers keep the
pucks positioned with identification
pods accessible for reading. The pucks
can then be locked in position with a
rod.
3. Testing
Manual handling of the SPINEplus,
miniSPINE and NewPin sample holders
as well as of the related manipulation
tools has been tested during the devel-
opment phase. Once selected for the
first implementation of a future sample-
holder standard, the miniSPINE system
was tested at 12 different partner sites
using evaluation kits (Supplementary
Fig. S5). Its design was then upgraded
following the feedback received. In
parallel, all of the models have been
tested under real conditions (crystal
harvesting, automated sample mounting
and data collection) at the ESRF–
EMBL–India BM14 beamline using a
FlexED8 sample changer (Papp et al.,
2017).
4. Results
Here, we only report on the manual
usability of the sample holders. The
results related to robotic sample trans-
fers are published in the accompanying
article (Papp et al., 2017) as they depend
on the robot grippers, the transfer times
and the precision of the robotics used.
The SPINEplus sample holders were
found to be easy to manipulate as they
are identical in size to the sample
holders commonly used at beamlines.
The miniSPINE harvesting tool was
judged to be convenient. However,
owing to the angle between the pin and
the tool, some users found it more
difficult to use than the usual straight
tools that are used to handle the SPINE sample holders where
the orientation of the loop can be easily selected. A major
problem was encountered with NewPin and miniSPINE pucks
when inserting the pins with a crystal mounted on them into a
puck under liquid nitrogen. Owing to the small size of the
wells, it can be difficult to know whether a position is occupied
or free. An additional difficulty when using NewPin was to
properly orient the pins to plug them correctly into the puck
slots. This made it clear that NewPin should be reserved for
fully robotic systems, including the crystal-harvesting step. For
miniSPINE, the puck-loading assistant (Fig. 4c) greatly
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Acta Cryst. (2017). D73, 829–840 Papp et al. A compact and precise sample holder 837
Figure 7
Sample-holder length compatibility. (a) Typical SPINE goniometer mount. (b) Positions of a
SPINE/SPINEplus pin, (c) of a miniSPINE pin and (d) of a NewPin pin mounted on a goniometer
socket.
Figure 8
Puck-handling tool and canisters. (a) Puck-handling tool grabbing a puck with its cover and
releasing the puck while keeping the cover, also showing a cut view of the locking mechanism. (b)
Eight-puck top-access canister. (c) Seven-puck shelved canister with locking rod, antirotation
fingers and apertures for manual gripper.
improved the manual harvesting process (Supplementary
Video S1).
5. Discussion
Two new sample holders for macromolecular cryocrystallo-
graphy have been developed together with corresponding
pucks and robotic and manual handling tools to enhance
crystal-processing times at beamlines and to reduce the
handling effort and transportation costs. miniSPINE allows
the storage of 36 samples in a puck. NewPin offers a storage
density of up to 64 samples per puck combined with highly
accurate positioning on goniometers. The two models can be
handled with the same robot gripper and use pucks with the
same footprint. They fit in a specific uni-puck-compliant dewar
slot and in canisters compatible with CX100 shipping dewars.
A goniometer mount compatible with SPINE and miniSPINE
was also developed to facilitate the integration of miniSPINE
at beamlines. The NewPin model requires a specific gonio-
meter mount and is more difficult to handle manually. It is
adapted to fully automated pipelines covering all of the steps
from crystal harvesting to processing at beamlines. The
maximum storage density of the NewPin pins could potentially
exceed 64 pins per puck as it depends on the space allocated
around the pins for handling. This is related to the outer
dimensions of the gripper and to the precision of the handling
robotics. Finally, a modified SPINE sample holder called
SPINEplus, which is backwards-compatible with SPINE, was
developed together with a miniSPINE/NewPin dewar slot
compliant puck. This interoperable line of sample holders and
pucks should facilitate the transition from the current SPINE
sample-holder standard to miniSPINE and further to NewPin,
in particular on beamlines that are equipped with flexible
sample changers based on six-axis industrial robots and tool
changers. Initially, sample tracking was identified as highly
important when moving to the densities proposed here;
therefore, both pins and pucks are ready to receive RFID tags.
Work is continuing to improve the reliability of the RFID tags
used to identify the pins so that they can resist the extreme
thermal cycling experienced during their lifetime. The identi-
fication of the pucks also includes Datamatrix and human-
readable labels. Defined as an important feature at the
beginning of the project, the identification of the pins was over
time judged to be less important than the identification of
pucks. In practice, even when available, SPINE-standard pin
identification is rarely used, probably because of the addi-
tional effort needed during crystal harvesting. Currently,
sample tracking almost exclusively relies on the position of the
pins in human-identifiable pucks. Furthermore, as automation
becomes more extensive, the potential for human error
decreases. In the near future, projects where individual sample
tracking is important should benefit from automated
harvesting and storing, making pin identification unnecessary.
The first pin and puck prototypes manufactured demon-
strated that the main drawback of increasing density is the
difficulty in manually handling the pins. A number of tools and
assistants were developed to facilitate crystal harvesting and
the storage of the pins in the pucks. The feedback received
from partner institutes and companies on the miniSPINE
manual handling tools were integrated into the present design.
Nevertheless, some limitations remain with the harvesting
tool, where the pins being set at 45(Figs. 2, 4 and 6) can
reduce the ability of the user to see and manipulate the
crystals in trays during harvesting. We should however point
out that the main goal of this work is to propose new sample
holders and that all of the related devices have essentially
been developed to assess the possibility of their manual
handling. As with existing sample holders, further tools can
develop over time. The second important aspect considered
was integration at beamlines. The main reason for miniSPINE
being proposed for the initial implementation of a new
sample-holder standard is the potential to operate a beamline
with both SPINE and miniSPINE, and because manual
handling is easier than with NewPin. NewPin is therefore
considered as a second implementation phase, or for a highly
demanding fully integrated platform from crystallization to
data collection. While sample turnover could be dramatically
increased at most recent third-generation synchrotron beam-
lines, new data-collection methods such as in situ data
collection and serial crystallography (Bingel-Erlenmeyer et al.,
2011; Axford et al., 2012; Gati et al., 2014; Zander et al., 2015)
disrupt the relationship between crystal and sample holder,
making future requirements unclear. Similarly, the emergence
of XFELs and the upgrade plans of many synchrotrons
worldwide are making sample delivery more and more
diversified (Lyubimov et al., 2015; Oghbaey et al., 2016; Suga-
hara et al., 2015; Weierstall et al., 2014), and therefore the
requirements for future sample holders are even less
predictable. Nevertheless, the new sample holders proposed
here should reduce the overall sample-handling efforts and
costs, as well as accelerating the alignment of crystals or areas
to scan at beamlines. This will be particularly true when they
are connected to future automated harvesting systems that are
anticipated to register crystal coordinates with individual pins.
Medium-size batches of the different sample holders and
the associated pucks have been manufactured for testing and
attempts have been made to find manufacturers able to
produce them in large quantities and at affordable cost. Key
companies working in the field of consumables for MX have
been associated with the project at an early stage to prepare
for the commercialization of the hardware involved. For large
production batches, the end price of the pins without RFID
tags should be comparable to the price of the current pins
(batches larger than 5000 units) and the end price of the pucks
expressed in cost per pin stored should be lower than the
average price of current pucks (batches larger than 100 units).
Moving from this feasibility study to a widely approved
standard is now the next important step. Knowing that the
production costs are largely dependent on the quantity
produced, and for NewPin on the investments made in tooling,
one of the main difficulties is to have affordable consumables
available for the pilot test sites. At the time of writing,
beamlines are hesitant to upgrade their robotics before
consumables are available, especially as many synchrotron
research papers
838 Papp et al. A compact and precise sample holder Acta Cryst. (2017). D73, 829–840
sites have already invested in large-capacity dewars to cope
with increasing sample turnover, currently making sample-
storage density a less critical issue. Therefore, the deployment
of miniSPINE will start at a limited number of, mostly
European, pilot beamlines, in parallel with the production of
the first batches of consumables.
The new sample supports presented here represent a new
opportunity for MX experiments where automation is playing
an ever-increasing role in seeking high reproducibility and
high throughput. It is hoped that miniSPINE, and eventually
NewPin, will play a central part in the future of ‘gene to
structure’ and in more sophisticated drug-development pipe-
lines.
Acknowledgements
We thank the BioStruct-X partners for their advice during the
project and their precious feedback on testing: Vincent
Olieric, Isabelle Martiel and Takashi Tomizaki at SLS (Paul
Scherrer Institute, Villigen, Switzerland), Uwe Mu
¨ller and
Manfred S. Weiss at BESSY II (Helmholtz-Zentrum Berlin fu
¨r
Materialien und Energie, Berlin, Germany), Marjolein
Thunnissen, Thomas Ursby and Uwe Mu
¨ller at MAX-IV
Laboratory (Lund, Sweden), Thomas Schneider, Gleb Bour-
enkov and Stefan Fiedler at EMBL@PETRA-III (European
Molecular Biology Laboratory, Hamburg, Germany), Sean
McSweeney, Didier Nurizzo and Christoph Mueller-Dick-
mann at ESRF (Grenoble, France), Katherine McAuley and
David Hall at Diamond Light Source Ltd (Oxford, England)
and the EMBL Grenoble Outstation (European Molecular
Biology Laboratory, Grenoble, France). We are grateful to the
other synchrotrons and beamlines that supported the NewPin
project: Sean McSweeney, Jean Jakoncic and Dieter Schneider
at NSLSII (Brookhaven National Laboratory, Upton, USA),
Xavier Vernede and Jean-Luc Ferrer at the IBS/CEA FIP-
BM30A/ESRF beamline (Grenoble, France), Alke Meents
and Nicolas Stuebe at PETRA-III (HASYLAB, DESY,
Hamburg, Germany) and Ueno Go at Spring-8 (Japan). We
thank Ulrich Zander at the European Molecular Biology
Laboratory, Grenoble, France for testing the sample holders.
This project was supported by the companies MiTeGen
(Ithaca, USA), Molecular Dimensions (Suffolk, England),
Irelec-Alcen (Saint-Martin-d’He
`res, France) and NatX-ray
(Saint Martin d’He
`res, France). We finally thank EMBLEM
Technology Transfer (Heidelberg, Germany) for managing the
legal and commercial aspects of this project. The authors
declare the following competing financial interest: the miniS-
PINE sample holder and puck are commercialized by
MiTeGen (Ithaca, USA) and Molecular Dimensions (Suffolk,
England) under EMBLEM licenses (Heidelberg, Germany).
Funding information
The research leading to these results has received funding
from the European Union Seventh Framework Programme
(FP7/2007–2013) under grant agreement BioStruct-X No.
283570.
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840 Papp et al. A compact and precise sample holder Acta Cryst. (2017). D73, 829–840
... More recently, developments in the field of sample mounts were mainly focused on standardizing the mounts with the aim of increasing sample throughput 16 or on designing mounts, which can hold more than one sample 17 , such as for instance patterned membranes on a silicon frame, which are capable of holding hundreds of small crystals mostly in the field of serial crystallography 18,19,20,21,22 . ...
... Various types of sample holders have been developed and are widely used in protein X-ray crystallography [1,2]. Collection of diffraction data from a single protein crystal involves a number of manual handling step, including crystal fishing, cryo protection and mounting onto an individual sample holder. ...
... Nevertheless, the size of the scan area should take into account the positioning discrepancy of the SPINE sample holder when mounted on the harvester and on the goniometer. This discrepancy can be reduced using NewPin sample holders [15], which provide a repositioning precision of 10 microns. Work is also ongoing to increase the speed of the calculation of the data collection strategy, and to remove all the dead time currently present in the execution of automated pipelines. ...
Conference Paper
CrystalDirect is a fully automated crystal harvesting system currently operating at the EMBL Grenoble and Hamburg outstations. The CrystalDirect harvester automatically harvests, cryo-cools and mounts on sample holders, crystals grown on an ultra-thin film directly compatible with X-ray data collection. Here, we report on CrystalDirect-to-Beam, a proof of concept made at the ESRF beamline ID30B, where a CrystalDirect harvester was coupled to the sample changer and diffractometer of the beamline. In this setup, pre-identified crystals provided in CrystalDirect crystallization plates are automatically harvested and directly transferred to the goniometer for X-ray data collection at cryogenic or room temperature. Crystal harvestings and data collections in cryo conditions have been automatically operated in a pipeline with model proteins, with results similar to those obtained with traditional methods. Experiments conducted at room temperature using a crystal dehydration device have shown that it is possible to harvest crystals and collect data at room temperature in optimal background conditions without manual intervention, and to automate time consuming crystal dehydration experiments. With CrystalDirect-to-Beam, we propose a new and flexible way to deliver crystals at beamlines, which minimizes sample handling and shortens crystal to data turnaround.
Article
Full-text available
An alternative storage method to separate sample preparation from single-crystal and powder X-ray diffraction measurements at home source diffractometers is described. For single crystals, a setup is presented which allows storage of preselected crystals under cryogenic and ambient temperatures. For powders, a disposable sample holder is introduced. The method is suitable for the storage of air- and moisture-sensitive samples. Equipment made of biodegradable polylactic acid is produced by 3D printing and can be adapted to individual needs. As 3D printers are widely available at research institutions nowadays, models of the presented equipment are provided for the reader to allow easy reproduction.
Article
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Here we present two robotic sample changers integrated into the experimental stations for the macromolecular crystallography (MX) beamlines AMX and FMX, and the biological small-angle scattering (bioSAXS) beamline LiX. They enable fully automated unattended data collection and remote access to the beamlines. The system designs incorporate high-throughput, versatility, high-capacity, resource sharing and robustness. All systems are centered around a six-axis industrial robotic arm coupled with a force torque sensor and in-house end effectors (grippers). They have the same software architecture and the facility standard EPICS-based BEAST alarm system. The MX system is compatible with SPINE bases and Unipucks. It comprises a liquid nitrogen dewar holding 384 samples (24 Unipucks) and a stay-cold gripper, and utilizes machine vision software to track the sample during operations and to calculate the final mount position on the goniometer. The bioSAXS system has an in-house engineered sample storage unit that can hold up to 360 samples (20 sample holders) which keeps samples at a user-set temperature (277 K to 300 K). The MX systems were deployed in early 2017 and the bioSAXS system in early 2019.
Article
Full-text available
Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425 , 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493 , 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015 , 1741 , 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5–30 keV photon energy range and delivers a flux of 4.0 × 10 ¹² photons s ⁻¹ at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 10 ¹² photons s ⁻¹ at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods – serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25 , 1362–1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ , 5 , 238–246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5 , 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.
Article
In this paper, the design and functionalities of the high-throughput TELL sample exchange system for macromolecular crystallography is presented. TELL was developed at the Paul Scherrer Institute with a focus on speed, storage capacity and reliability to serve the three macromolecular crystallography beamlines of the Swiss Light Source, as well as the SwissMX instrument at SwissFEL.
Article
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Reducing the sample-exchange time is a crucial issue in maximizing the throughput of macromolecular crystallography (MX) beamlines because the diffraction data collection itself is completed within a minute in the era of pixel-array detectors. To this end, an upgraded sample changer, SPACE-II, has been developed on the basis of the previous model, SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), at the BL41XU beamline at SPring-8. SPACE-II achieves one sample-exchange step within 16 s, of which its action accounts for only 11 s, because of three features: (i) the implementation of twin arms that enable samples to be exchanged in one cycle of mount-arm action, (ii) the implementation of long-stroke mount arms that allow samples to be exchanged without withdrawal of the detector and (iii) the use of a fast-moving translation and rotation stage for the mount arms. By pre-holding the next sample prior to the sample-exchange sequence, the time was further decreased to 11 s in the case of automatic data collection, of which the action of SPACE-II accounted for 8 s. Moreover, the sample capacity was expanded from four to eight Uni-Pucks. The performance of SPACE-II has been demonstrated in over two years of operation at BL41XU; the average number of samples mounted on the diffractometer in one day was increased from 132 to 185, with an error rate of 0.089%, which counted incidents in which users could not continue with an experiment without recovery work by entering the experimental hutch. On the basis of these results, SPACE-II has been installed at three other MX beamlines at SPring-8 as of July 2019. The fast and highly reliable SPACE-II is now one of the most important pieces of infrastructure for the MX beamlines at SPring-8, providing users with the opportunity to fully make use of limited beamtime with brilliant X-rays.
Article
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ID30B is an undulator-based high-intensity, energy-tuneable (6.0–20 keV) and variable-focus (20–200 µm in diameter) macromolecular crystallography (MX) beamline at the ESRF. It was the last of the ESRF Structural Biology Group's beamlines to be constructed and commissioned as part of the ESRF's Phase I Upgrade Program and has been in user operation since June 2015. Both a modified microdiffractometer (MD2S) incorporating an in situ plate screening capability and a new flexible sample changer (the FlexHCD) were specifically developed for ID30B. Here, the authors provide the current beamline characteristics and detail how different types of MX experiments can be performed on ID30B (http://www.esrf.eu/id30b).
Article
Full-text available
Automated sample changers are now standard equipment for modern macromolecular crystallography synchrotron beamlines. Nevertheless, most are only compatible with a single type of sample holder and puck. Recent work aimed at reducing sample-handling efforts and crystal-alignment times at beamlines has resulted in a new generation of compact and precise sample holders for cryocrystallography: miniSPINE and NewPin [see the companion paper by Papp et al. (2017, Acta Cryst. , D 73 , doi:10.1107/S2059798317013742)]. With full data collection now possible within seconds at most advanced beamlines, and future fourth-generation synchrotron sources promising to extract data in a few tens of milliseconds, the time taken to mount and centre a sample is rate-limiting. In this context, a versatile and fast sample changer, FlexED8, has been developed that is compatible with the highly successful SPINE sample holder and with the miniSPINE and NewPin sample holders. Based on a six-axis industrial robot, FlexED8 is equipped with a tool changer and includes a novel open sample-storage dewar with a built-in ice-filtering system. With seven versatile puck slots, it can hold up to 112 SPINE sample holders in uni-pucks, or 252 miniSPINE or NewPin sample holders, with 36 samples per puck. Additionally, a double gripper, compatible with the SPINE sample holders and uni-pucks, allows a reduction in the sample-exchange time from 40 s, the typical time with a standard single gripper, to less than 5 s. Computer vision-based sample-transfer monitoring, sophisticated error handling and automatic error-recovery procedures ensure high reliability. The FlexED8 sample changer has been successfully tested under real conditions on a beamline.
Article
Full-text available
Automated sample changers are now standard equipment for modern macromolecular crystallography synchrotron beamlines. Nevertheless, most are only compatible with a single type of sample holder and puck. Recent work aimed at reducing sample-handling efforts and crystal-alignment times at beamlines has resulted in a new generation of compact and precise sample holders for cryocrystallography: miniSPINE and NewPin [see the companion paper by Papp et al. (2017, Acta Cryst. , D 73 , doi:10.1107/S2059798317013742)]. With full data collection now possible within seconds at most advanced beamlines, and future fourth-generation synchrotron sources promising to extract data in a few tens of milliseconds, the time taken to mount and centre a sample is rate-limiting. In this context, a versatile and fast sample changer, FlexED8, has been developed that is compatible with the highly successful SPINE sample holder and with the miniSPINE and NewPin sample holders. Based on a six-axis industrial robot, FlexED8 is equipped with a tool changer and includes a novel open sample-storage dewar with a built-in ice-filtering system. With seven versatile puck slots, it can hold up to 112 SPINE sample holders in uni-pucks, or 252 miniSPINE or NewPin sample holders, with 36 samples per puck. Additionally, a double gripper, compatible with the SPINE sample holders and uni-pucks, allows a reduction in the sample-exchange time from 40 s, the typical time with a standard single gripper, to less than 5 s. Computer vision-based sample-transfer monitoring, sophisticated error handling and automatic error-recovery procedures ensure high reliability. The FlexED8 sample changer has been successfully tested under real conditions on a beamline.
Article
Full-text available
The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine φ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed.
Article
Full-text available
Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF-1 (ID30A-1), based on an industrial six-axis robot, is described. The device, named RoboDiff, includes a high-capacity dewar, acts as both a sample changer and a high-accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF-1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically.
Article
Full-text available
The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.
Article
Full-text available
Following pioneering work 40 years ago, synchrotron beamlines dedicated to macromolecular crystallography (MX) have improved in almost every aspect as instrumentation has evolved. Beam sizes and crystal dimensions are now on the single micron scale while data can be collected from proteins with molecular weights over 10 MDa and from crystals with unit cell dimensions over 1000 Å. Furthermore it is possible to collect a complete data set in seconds, and obtain the resulting structure in minutes. The impact of MX synchrotron beamlines and their evolution is reflected in their scientific output, and MX is now the method of choice for a variety of aims from ligand binding to structure determination of membrane proteins, viruses and ribosomes, resulting in a much deeper understanding of the machinery of life. A main driving force of beamline evolution have been advances in almost every aspect of the instrumentation comprising a synchrotron beamline. In this review we aim to provide an overview of the current status of instrumentation at modern MX experiments. The most critical optical components are discussed, as are aspects of endstation design, sample delivery, visualization and positioning, the sample environment, beam shaping, detectors and data acquisition and processing.
Article
Full-text available
Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities.
Article
Full-text available
The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. Sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade.
Article
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Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacteriorhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.
Article
Full-text available
MASSIF-1 (ID30A-1) is an ESRF undulator beamline operating at a fixed wavelength of 0.969 Å (12.8 keV) that is dedicated to the completely automatic characterization of and data collection from crystals of biological macromolecules. The first of the ESRF Upgrade MASSIF beamlines to be commissioned, it has been open since September 2014, providing a unique automated data collection service to academic and industrial users. Here, the beamline characteristics and details of the new service are outlined.
Article
Automation is beginning to transform the way data are collected in almost all scientific disciplines. The combination of robotics and software now allows data to be collected consistently and reproducibly, eliminating human error and boredom. This approach has been applied to macromolecular crystallography at MASSIF-1, a fully automated beamline at the European Synchrotron Radiation Facility (ESRF). Considerable human effort is still dedicated to evaluating protein crystals in order to find the few crystals that diffract well or collecting hundreds of data sets to screen potential new drug candidates. The combination of ESRF-developed robotic sample handling and advanced software protocols now provides a new tool to structural biologists. Not only is the beamline used efficiently, running 24 h a day without getting tired, data collection is also performed consistently by an expert system, often better than with a human operator. In this review, we will focus on the impact this level of automation has had on the optimum acquisition of data from crystals of biological macromolecules.