CM01 Cryo-electron microscope

LOGO CM01 Bleu.jpg (LOGO CRYO_LOGO CM01 Bleu)

 

 

Synopsis

The ESRF has acquired a Titan Krios cryo-electron microscope (cryoEM) for single particle experiments. It is equipped with a K2 Summit direct electron detector, a Quantum LS imaging filter and a Volta phase plate (VPP). The microscope is situated at the ESRF and is run as a beamline (CM01) with access for the international Structural Biology community given on scientific merit through rolling-access applications. In the longer term, CM01 will be  part of a cryoEM platform run within the Partnership for Structural Biology (PSB) in conjunction with the microscopes located at the IBS and the EMBL. A team of three scientists from the ESRF, the IBS and the EMBL will support user operation.

Status: full user operation

Operation

Access will be given through rolling-access applications which is open since September 2017. Initially, a minimum of 9 shifts per experiment will be scheduled.

Location

Microscope CM01 is located in the ESRF Belledonne experimental hall between beamlines ID01 and ID02.

CM01 location in Belledonne exprimental hall extension  

 

Use of Cryo-EM

Cryo-EM can be used for protein structure determination with near atomic resolution by single-particle imaging. At the ESRF, Cryo-EM is a complementary technique to macromolecular crystallography and BioSAXS. For a protein that can be crystallised, macromolecular X-ray diffraction (MX) is still the most efficient means to obtain its structure at very high resolution. In contrast, BioSAXS provides low resolution details about the overall shape of a single protein or a macromolecular complex in solution. Cryo-EM has been used to obtain subnanometre-resolution structures of up to ~2 Å for ideal structures including protein complexes and viruses with sizes typically of 150 kDa and above [1]. It is commonly used for larger proteins and complexes that are intrinsically difficult to crystallise.  Cryo-EM image analysis is powerful enough to enable in silico classification of different conformation states of the sample if they co-exist in solution.

Sample preparation

During the early stage of operation (until end 2018), only frozen and pre-characterised samples will be accepted for experiments using the Titan Krios microscope. Proof must be included with the proposal (Cryo-EM raw images satisfying class averages and/or a first 3D reconstruction), to justify the use of this high-end microscope.

An experiment

Cryo-EM data collection and analysis takes considerably longer than an X-ray diffraction experiment. Expect to screen only a few sample grids and collect data on one grid (or only a few grids) during a user session. Slots for data collection are allocated as multiples of 3 eight-hour shifts (i.e. a 24-hour day) depending on the experimental needs. At the beginning, 3 days will be allocated per experiment. Images will be collected and a first analysis (CTF estimation) will be done.

User’s data can be saved to external hard disks using a dedicated data backup PC in the control cabin. Dedicated computing power for image analysis will be provided at a later stage. Please remember to come with a hard disk drive of sufficiently large size (we recommend a USB3 connection and a minimum of 4 TB).

Applying for beamtime

Applications to use microscope CM01 are made through the ESRF User Portal (SMIS). The type of proposal is a structural biology rolling-access application, which can be submitted at any time during the year. For accepted proposals, travel expenses will be reimbursed for a maximum of two visitors per experiment.

Sufficient proof of the sample’s quality must be included with the proposal. While the written part of an application is limited to a maximum of two pages, evidence of the sample’s quality can be provided in an additional document, also limited to two pages.

The voltage required for the experiment should be noted in the rolling proposal tab "Beamline(s) requested” in the field “Other”, situated at the bottom of the beam requirements section. But please note that, in the beginning, all experiments will be carried out at 300 kV.
In the "Sample Description" tab, the molecular mass of your macromolecule or complex should be indicated, and please describe the grid (type, hole size, etc.) in the "container" field.

Click here to see the obligatory fields to be filled for the proposal form.

Titan Krios specifications

Accelerating voltage

300 kV (lower accelerating voltages will be made available at a later stage).

Electron source

High brightness Schottky Field Emission Gun (X-FEG).

Lens system

3 condenser lens system.
C-TWIN objective lens (focal length = 3.5 mm; Cc = 2.7 mm; Cs = 2.7 mm) allows for tilts up to 70°.

Operating temperature

100°K

Sample handling system

Cryo-autoloader that can accommodate a maximum of twelve AutoGridsTM.

Sample exchange time

< 1 min if in the cryo-autoloader.

Detector

Gatan K2 summit direct electron detector mounted on a Gatan Bioquantum LS/967 energy filter. Counting or super resolution mode (8 k x 8 k).

Phase plate

FEI Volta phase plate allowing phase shift tuneable between 45° and 90° to provide optimal phase transfer.

Acquisition software

Latest version of EPU.
Other systems may be implemented later.

References

[1] Breaking cryo-EM resolution barriers to facilitate drug discovery, A. Merk, A. Bartesaghi, S. Banerjee, V. Falconieri, P. Rao, M.I. Davis, R. Pragani, M.B. Boxer, L.A. Earl, J.L.S. Milne, and S. Subramaniam, Cell 165, 1698–1707 (2016); doi: 10.1016/j.cell.2016.05.040.