EMBL@XFEL Questionnaire

The European Molecular Biology Laboratory (EMBL) and the European X-ray Free Electron Laser (XFEL) at DESY, Hamburg, Germany, have a keen interest in the development of FEL-based applications in structural biology, making full usage of the new FEL light sources, and in the development of state-of-the-art instrumentation to enhance the potential results of their scientific exploitation. For a brief introduction as to the possibilities for biological experiments at XFEL the reader is referred here.

A formal framework has been established by a Memorandum of Understanding between the EMBL and the XFEL signed in September 2011. Within this framework the EMBL, together with international collaborators, proposes to construct a biology infrastructure at the XFEL facility (expected completion in 2016) to enable biological experiments using the high brilliance, ultrashort pulse duration and high repetition rate coherent X-ray radiation. This facility is foreseen to be an integrated part of the current or forth-coming biological infrastructures at the DESY campus.

The proposed biological sample infrastructure facility will provide:

support to the international life-science community in generation and handling of challenging samples in immediate proximity to XFEL instruments.
appropriate selection, quality control and evaluation of samples, including correlative imaging, immediately prior to XFEL experiments.
standardized technology for data interpretation, including computation and validation of structural models.

The biology infrastructure will specifically target and provide novel opportunities in diffractive imaging of:

crystals (or other repetitive objects) on a micro- or nano-scale.
single particles, including approximately identical (eg. protein complexes) or different objects (organelles or whole cells).

Current expectations are that nano-crystallography will provide high-resolution diffraction data sets using serial crystallography from small (sub-micron) samples. Coherent X-ray imaging of identical or non-identical single-particle objects will allow the collection of multiple 2D projections, which may then be reassembled into a high-resolution (1-10 nm) image of the sample (large macromolecular complexes, organelles and whole cells).

In order to ensure efficient operation of the facility and provide optimal services for the international biological community we seek your input to identify the requirements necessary to support these novel biological imaging experiments.

The results of this survey will be used to identify the most pressing needs of the international community that should be taken into account during planning and construction of the infrastructure.

Your details

Your name and email address will be used to contact you for more information (if needed).

Your name
Your email address
Your institute
Your field of research

Please rank the following in terms of their importance to any future research that you would intend to carry out at XFEL (1 - not important; 2 - could be useful, 3 - required, 4 - important, 5 - essential, 6 - no opinion).

Types of experiments envisaged

Single particle imaging of biomolecules and macromolecular complexes

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Identical, nonperiodic: dynamics of macromolecular complex formation
Non-identical, periodic: filaments, viruses
Non-identical, non-periodic: organelles or cells

Nano-crystallography

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Identical, periodic samples: serial crystallography on nano-micron-sized macromolecular crystals

Time-resolved Studies

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Pump-probe (single particles)
Pump-probe (crystals)
time regime of interest

Other types of experiments

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6

please specify type

Single particle imaging experiments

Sample production (large protein complexes, cell lines, etc.)

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Bacterial culturing facilities (E. coli, other)
please specify type

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Eukaryotic culturing facilites (e.g yeast, insect cell, HeLa, CHO, HEK 293, other)
please specify type

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Cell-free expression (E. coli lysate, wheat germ lysate)
please specify type

Sample purification (large protein complexes)

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Micro-scale purification (µg-pg)
Macro-scale purification (mg-µg)
Affinity tagged (His/GST/etc.)
Storage for samples purified externally
Other
please specify

Sample storage

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Room temperature
4°C incubators
-20°C refrigerators
-80°C refrigerators
LN₂ storage
Storage for samples produced internally
Storage for samples produced externally
Other
please specify

Quality control and analytical methods

(NB. Some of these may be offered by collaborative institutes within the DESY campus, rank highest those you feel should be sited at the XFEL instrument)

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Dynamic light scattering
Static light scattering
SDS-PAGE (incl. Western Blotting)
Mass spectrometry (non-denaturing: e.g. confirmation of protein complex fidelity and/or sample identification)
please specify type
Electron microscopy
Scanning electron microscopy
Transmission electron microscopy
Cryo-electron microscopy
Electron tomography
Isothermal calorimetry
UV-Vis Absorption Spectroscopy
Light and fluorescence microscopy
Circular dichroism
Analytical ultracentrifugation
Comparative centrifugation
Surface plasmon resonance
Other
please specify

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Fluorimetry-based kinetic assays (thermofluor)
Fluorimetry-based kinetic assays (nano-drop)
Fluorimetry-based kinetic assays (plate-reader)
Fluorimetry-based kinetic assays (other)
please specify type

Nano-crystallography

Crystal growth

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Small-scale (nl) crystallization robotics
Large scale (µl-ml) crystallization robotics
2D crystallization
Optical microscopy
Remote imaging
Diffraction screening possibilities prior to XFEL analysis
Other
please specify
Storage for samples crystallized externally

Data collection method

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Fixed target (water or volatile buffers; sample is dried or cryo-frozen)
Liquid Jet (most physiological buffers)
Aerosol (volatile buffers)
Electrospray
Other
please specify
Remote data collection options

Data analysis

A single experiment may generate terabytes of data.

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Online data analysis (hit identification)
Offline data analysis
Remote data analysis
Provision of computational power (i.e. cluster)
Support in data processing
Data Processing as a service
Workshops on specific software

Biological Safety

The current plans are centered on the construction of an S1 facility. However, future demands may modify the design. Please indicate the maximum level of biological safety that your future experiments would require:

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Biological Safety Level: 1
Biological Safety Level: 2
please give examples of projects
Biological Safety Level: 3
please give examples of projects
Biological Safety Level: 4
please give examples of projects

General

Although experiments are anticipated to require relatively short periods of time, sample preparation may require more extended stays.

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
On-site training
On-site seminar series
Off-site training
Off-site seminars
Experimental Support (Sample production)
Experimental Support (Data collection)
Experimental Support (Data analysis)
Sample production (as service or collaboration)
Data collection (as service or collaboration)
Data analysis (as service or collaboration)
Remote access
Accommodation

Your anticipated involvement

Your anticipated involvement in setting up and/or operation of the biology infrastructure facility:

	(1 - not important; 5 - essential; 6 - no opinion)
	1	2	3	4	5	6
Access to the facility on a competitive basis (peer-reviewed applications)
please give examples of project types

and an estimate of the amount of samples you forsee to measure per annum
Collaboration
please specify or give examples
In-kind contribution of equipment or consumables
please specify or give examples
Provision of financial, technical, staff or other type of resources
please specify or give examples

Any additional comments

Please feel free to provide any other comments regarding equipment/installations you feel would be necessary or contributions that you might be able to provide:

Please address questions and comments about research infrastructure to Victor Lamzin.
Please report any technical problems with this questionnaire to Saul Hazledine.