CCP-EM Spring Symposium 2021 - Virtual Symposium held via Zoom Scientific Organisers: Giulia Zanetti (Birkbeck, University of London) Christopher ...
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CCP-EM Spring
Symposium 2021
Virtual Symposium held via Zoom
21st – 23rd April 2021
Scientific Organisers:
Giulia Zanetti (Birkbeck, University of London)
Christopher Aylett (Imperial College London)Abstracts
Misha Kudryashev (Max Planck Institute of Biophysics) Page
Structure of ion channels in membranes by cryo-EM 2
Danielle Paul (Bristol) Page
In situ cryo-electron tomography reveals filamentous actin with the microtubule lumen 3
Alberto Bartesaghi (Duke) Page
High-throughput, high-resolution structure determination using single-particle cryo-ET 4
Francesca Coscia (Human Technopole) Page
The structure of human thyroglobulin 5
Radoslav Enchev (Crick) Page
Visual biochemistry 6
Lori Passmore (MRC-LMB) Page
Molecular insights into gene expression 7
Peijun Zhang (DLS/Oxford) Page
Visualizing SARS-CoV-2 infection and vaccine by multi-scale cryo-imaging 8
James Krieger (CSIC) Page
ProDy 2.0 and CryoDy: Extension of protein dynamics analyses to large scale and cryo-EM maps 9
Takanori Nakane (MRC-LMB) Page
Single-particle cryo-EM at atomic resolution 10
Ellen Zhong (MIT) Page
Advances in heterogeneous reconstruction with cryoDRGN 11
Hans Elmlund (Monash) Page
SIMPLE 3.0: SPA in real time and new methods for motion correction of cryogenic and liquid samples 12
Matthias Wolf (OIST) Page
Beyond the potential: Cryo-TEM-EELS for bio samples 13
Mateusz Olek (DLS / York) Page
The IceBreaker software for automated estimation and removal of the ice gradient on cryo-EM micrographs 14
Grzegorz Chojnowski (EMBL) Page
Identification of unknown proteins and sequence assignment validation in cryo-EM 15
Carlos Oscar Sorzano (CSIC) Page
Bias and variance in CryoEM 16
Maya Topf (Birkbeck / CSSB Hamburg) Page
Assessment and refinement of models from CASP14 cryo-EM targets 17
1Misha Kudryashev
Max Planck Institute of Biophysics
Structure of ion channels in membranes by cryo-EM
Ion channels open and close ion-conductive pores in membranes regulating many
important physiological processes including synaptic transmission and propagation of
action potentials. Native lipids, transmembrane gradients, interacting partners
modulate structure and function of ion channels in physiological settings and are often
lost during the preparation of the channels for structural analysis. The established and
the developing methods of cryo-EM allow gaining unique insights into regulation of
membrane proteins by lipids.
During the talk I will demonstrate the impact that native lipids have on the structure of
ion channels using the giant calcium channel RyR1 from rabbit muscle as an example
[1,2]. I will furthermore discuss the challenges in determining higher resolution
structure of non-overwhelmingly abundant membrane proteins in situ by cryo electron
tomography and subtomogram averaging. The key issues are throughput of data
collection and processing as well as extraction of high-resolution information from cryo
electron tomograms. I will share the new developments from our group which are the
near-automated data processing workflow and a faster and arguably better alternative
to subtomogram averaging by sub-stack analysis [3].
Further reading:
1. Sanchez R, Zhang Y, Chen W, Dietrich L, Kudryashev M, Subnanometer-
resolution structure determination in situ by hybrid subtomogram averaging - single
particle cryo-EM. Nature Communications, 2020, 11: 3709.
2. Chen W, Kudryashev M, Structure of RyR1 in native membranes, EMBO Reports,
2020, 9:e49891
3. Sánchez RM, Mester R, Kudryashev M, Fast Cross Correlation for Limited Angle
Tomographic Data. In: Felsberg M., Forssén PE., Sintorn IM., Unger J. (eds) Image
Analysis. SCIA 2019. Lecture Notes in Computer Science, vol 11482. Springer. Doi:
10.23919/EUSIPCO.2019.8903041
2Danielle Paul
Bristol
In situ cryo-electron tomography reveals filamentous actin with the
microtubule lumen
Many cellular processes like migration and cell division depend on filamentous (F-)
actin and microtubules. These dynamic filamentous proteins are major components of
the cytoskeleton. Biophysical and cell biology experiments continue to reveal how the
actin and microtubule cytoskeletons work together and are intimately linked. The
complex interactions or crosstalk between these two structural scaffolds is thought to
be regulated by proteins that interface between them. The role of many microtubule
associated proteins (MAPs) is now becoming clearer, however less is known about
the role of proteins that reside within the microtubule lumen. Microtubules assemble
from tubulin dimers to form hollow tubes of protofilaments with a luminal width of
~15nm, which limits access to antibody epitope or small -molecule binding sites, used
in the detection of associated proteins. We have used cryo-electron tomography to
demonstrate that the microtubule lumen can be occupied by extended segments of F-
actin in small-molecule induced, microtubule-based cellular projections [1]. This is the
first observation of these two crucial protein scaffolds in such a conformation. We
determined two classes of actin filament and evidence of an additional protein
regularly labelling the actin in the class II filaments.
We have uncovered an unexpected versatility in cytoskeletal form that may prompt a
significant development of our current models of cellular architecture and offer a new
experimental approach for the in situ study of microtubule structure and contents.
3Alberto Bartesaghi
Duke
High-throughput, high-resolution structure determination using
single-particle cryo-ET
Tomographic reconstruction of cryo-preserved specimens followed by extraction and
averaging of sub-volumes has been successfully used to determine the structure of
macromolecules in their native environment.
Eliminating biochemical isolation steps required by other techniques, this method
opens up the cell to in-situ structural studies. Delays introduced during mechanical
navigation of the specimen and stage tilting, however, significantly slow down data
collection and the lower contrast and quality of tilted projections limits the resolution of
3D reconstructions. Here, I present BISECT (Beam Image-Shift Electron Cryo-
Tomography), a protocol to accelerate tilt-series acquisition without sacrificing
resolution that uses beam-image shift to multiply the number of areas imaged at each
stage position. I also describe our new CSPT (Constrained Single Particle
Tomography) data processing framework that significantly improves reconstruction
quality by performing per-tilt astigmatic CTF estimation and self-tuning exposure
weighting. For validation, we collected tilt-series from a low molecular weight target
(~300kDa) in parallel mode and obtained a map at near-atomic resolution. These
advances bring cryo-ET a step closer to becoming a high-throughput technique for in-
situ structure determination at high-resolution.
4Francesca Coscia
(Human Technopole)
The structure of human thyroglobulin
Thyroglobulin (TG) is a large dimeric glycoprotein (600 kDa) and precursor of thyroid
hormones (TH), essential activators of metabolism in vertebrates. TH synthesis from
TG occurs in the thyroid gland via the iodination and coupling of proximal tyrosine
pairs. Interestingly, out of about 60 iodinated tyrosines within TG, only a few can
generate TH, while the rest serves as iodine reservoir for the organism. How the TG
structure carves hormonogenic sites and how key tyrosines originate TH, have been
outstanding questions in thyroid biology. We determined the first and full-length
structure of human thyroglobulin at 3.5 Å by single particle electron cryo-microscopy
(cryo-EM). An initial C2 reconstruction produced a map of the elongated TG dimer,
which was anisotropic in resolution. A combination of symmetry expansion and
focused refinement led us to a high-quality map suitable for de novo model building.
93% of the protein sequence and several glycosylation sites important for the
intertwined TG dimer stability were resolved. From the structure we could uniquely
identify - and biochemically verify - a stoichiometry of seven TH formed per TG dimer,
from as many sites. Surprisingly, the reactive tyrosine pairs are loosely attached to TG
and not in a geometrically or chemically constrained active site. Proximity, flexibility,
solvent accessibility of tyrosine pairs appear to be the only determinants for efficient
coupling leading to TH synthesis. To recapitulate this mechanism, we engineered
flexible tyrosine pairs in an unrelated bacterial protein and obtained TH production with
an efficiency comparable to that of TG. This structure provides an important framework
to interpret TG mutations related to widespread thyroid diseases. The role of the
evolutionary conserved TG dimeric structure in TH homeostasis remains to be
elucidated. In this direction, this study opens new horizons for future mechanistic
investigations of TH synthesis regulation across species.
5Radoslav Enchev
Crick
Visual biochemistry
6Lori Passmore
MRC-LMB
Molecular insights into gene expression
7Peijun Zhang
DLS/Oxford
Visualizing SARS-CoV-2 infection and vaccine by multi-scale cryo-
imaging
Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural
studies on purified recombinant viral components and inactivated viruses. However,
structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses
in the frozen-hydrated native cellular context is scarce, and there is a lack of
comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the
cytopathic events induced by SARS-CoV-2 with virus replication process under the
frozen-hydrated condition, here we established a multi-modal, multi-scale cryo-
correlative platform to image SARS-CoV-2 infection in Vero cells. This platform
combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with
cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging.
The results place critical SARS-CoV-2 structural events – e.g. viral RNA transport
portals on double membrane vesicles, virus assembly and budding intermediates,
virus egress pathways, and native virus spike structures from intracellular assembled
and extracellular released virus - in the context of whole-cell images. This integrated
approach allows a holistic view of SARS-CoV-2 infection process, from the whole cell
to individual molecules.
8James Krieger
CSIC
ProDy 2.0 and CryoDy: Extension of protein dynamics analyses to
large scale and cryo-EM maps
Cryo-EM has the potential to produce structures of large and dynamic supramolecular
complexes that are not amenable to traditional approaches for studying structure and
dynamics. Their size and low resolution often make structural modelling and molecular
dynamics simulations challenging and computationally expensive. We therefore
developed a computational methodology based on elastic network models adapted to
cryo-EM density maps to gain a deeper understanding of their structure-encoded
allosteric dynamics. We applied this approach to the chaperonin TRiC/CCT, which
plays a major role in assisting the folding of many proteins through an ATP-driven
allosteric cycle and has recently gained a wealth of structural information from Cryo-
EM. Our analysis yielded a new pipeline in the ProDy Python application programming
interface for protein dynamics, incorporating the topology representing network
algorithm and hybrid simulation methods. Using these techniques together with
existing tools within ProDy, we analysed several structures of the chaperonin resolved
in different states toward mapping its conformational landscape and allosteric
mechanisms. Our study indicates that the overall architecture intrinsically favours
cooperative movements that comply with the structural variabilities observed in
experiments. Furthermore, the individual subunits CCT1-CCT8 exhibit state-
dependent sequential events at different states of the allosteric cycle. For example, in
the ATP-bound state, subunits CCT5 and CCT4 selectively initiate the lid closure
motions favoured by the overall architecture, whereas in the apo form of the
heteromer, the subunit CCT7 exhibits the highest predisposition to structural change.
The changes then propagate through parallel fluxes of allosteric signals to neighbours
on both rings. The predicted state-dependent mechanisms of sequential activation
provide new insights into TRiC/CCT intra- and inter-ring signal transduction events.
We will present these algorithmic developments and biological results together with
an overview of ProDy 2.0, which we have recently released including new ensemble
generation methods and the SignDy pipeline for large scale analyses of protein
families.
9Takanori Nakane
MRC-LMB
Single-particle cryo-EM at atomic resolution
Resolution we can obtain by cryo-EM single particle analysis of biological
macromolecules has steadily improved year by year. Although many biological
questions can be answered at mid-to-low resolutions currently available, chemical
understanding of enzymatic reaction mechanisms and protein-ligand interactions
requires higher resolutions that can precisely locate individual atoms including
hydrogens and solvents.
In 2020, two groups achieved the milestone of true atomic resolution.
Our team made the most of a new microscope developed by Thermo Fisher Scientific
equipped with a cold field emission gun (cold FEG), a stable energy filter (Selectris X)
and a faster direct electron detector (Falcon4) to solve the structure of apoferritin at
1.22 Å.
We also used this scope to resolve a human membrane protein, the β3 GABAA
receptor homopentamer at 1.7 Å.
In both cases, many ordered hydrogen atoms were visualized in difference maps.
I will explain how we achieved this resolution and discuss future research directions
to bring sub-2Å resolution to more targets and extract more information such as charge
distributions.
10Ellen Zhong
MIT
Advances in heterogeneous reconstruction with cryoDRGN
11Hans Elmlund
Monash
SIMPLE 3.0: SPA in real time and new methods for motion
correction of cryogenic and liquid samples
I will introduce the third major release of the SIMPLE (Single-particle IMage
Processing Linux Engine) open-source software package for analysis of cryo-EM
movies of single-particles (SPs). SIMPLE 3.0 enables real-time data processing using
minimal CPU computing resources to allow easy and cost-efficient scaling of
processing as data rates escalate and features an easy-to-use web-based GUI that
can be run on any device (workstation, laptop, tablet or phone), supporting a remote
multi-user environment over the network. Using SIMPLE 3.0, it is possible to
automatically obtain a clean SP data set amenable to high-resolution 3D
reconstruction directly upon completion of the data acquisition, without the need for
extensive image processing post collection. If time permits, I will introduce some of
our work in progress, which includes regularized anisotropic motion correction where
the deformation model is part of the objective function and thus smoothens the jittery
shift trajectories as part of the optimization process.
12Matthias Wolf
OIST
Beyond the potential: Cryo-TEM-EELS for bio samples
Biological cryo-EM has become a powerful technique for structure determination of
radiation-sensitive organic samples at near-native conditions. Its amazing success in
the past decade is often attributed to the advent of direct electron detection detectors
(DDD) and advances in the stability of modern electron microscopes as well as
computational image processing. Typically, protein and/or nucleic acids frozen in
amorphous ice are sampled by a charged particle beam, resulting in an image that
represents a projection of the electron potential of the three-dimensional (3D) object.
Single particle analysis allows determination of the orientations of these two-
dimensional particle images and reconstruction of a 3D electron potential map. This
3D-reconstruction process is based on the assumption that the imaged particle is a
thin-phase object, which only considers the amplitude contrast fraction as a simple
term in the phase contrast transfer function. Amplitude contrast is chiefly due to
inelastic scattering, which is associated with energy loss. The inelastic image is formed
by a different wavelength and is therefore either purposefully eliminated with an energy
filter (zero-loss filtering), or its contribution is generally ignored in the reconstruction
process. Material scientists have long known to exploit the information in the energy
loss spectrum with the purpose to identify the electronic signatures of elements and
hence to identify the atomic composition of the object both qualitatively and
quantitatively. However, because electron energy-loss spectroscopy (EELS) typically
requires high electron dose for adequate signal-to-noise ratio, it has mainly been
limited to inorganic radiation-hard samples such as metals or semiconductors. Direct
electron detectors can also greatly improve the image quality for spectroscopy at
medium to low electron dose. In this talk, I will present the basics of EELS and our
own results using a modified K2 DDD. I will demonstrate metal identification in
archaeal filaments and briefly discuss applications of EELS and energy-filtered TEM
with biological specimen.
13Mateusz Olek
DLS / York
The IceBreaker software for automated estimation and removal of
the ice gradient on cryo-EM micrographs
With many software tools available the optimal particle selection is still a vital issue in
the cryoEM. Regardless of the methods used, most pickers struggle when the varying
ice thickness is present on the micrograph. The proposed solution allows to estimate
the relative ice gradient and remove it, based on the K-Means clustering algorithm.
Used method results with micrographs which have the contrast equalized locally in
order to differentiate the particles from the background features, aiming to improve the
particle pickers performance. It can be used as a part of the data processing pipeline
to pick as many particles as possible from each micrograph after this pretreatment.
Additionally, a new parameter corresponding to the local ice thickness is introduced
for each of the picked particles. Based on this parameter, the picks can be grouped,
sorted and filtered during the next stages of processing. The presented software can
be also used as a validation tool for already refined maps, deposited with the
coordinates of the selected particles to asses how the particles from different ice
thickness areas contributed to the final map. Finally, the estimated ice gradient
distributions can be stored separately and used to inspect the general quality of
prepared samples.
14Grzegorz Chojnowski
EMBL
Identification of unknown proteins and sequence assignment
validation in cryo-EM
Although single particle cryo-EM usually targets known proteins, chains of unknown
sequence are often encountered. They can be purified from natural sources or appear
as an unexpected fragment of a well characterized protein. Regardless of the source
of the problem, the unknown protein always requires tedious characterization.
To address this issue we have developed a complete pipeline for the identification of
unknown proteins in cryo-EM reconstructions. A key element in the pipeline is a
machine-learning based computer program findMySequence that identifies the most
plausible protein sequence in a sequence database, given a cryo-EM reconstruction
and a main-chain only model. Our largescale benchmarks show that the program
successfully identifies sequences of protein backbone models automatically built into
cryo-EM maps at local resolution as low as 4.5 Å, which are usually highly fragmented
and prone to tracing errors.
We also show that the method can be used for detecting sequence assignment errors
in cryoEM. Indeed, our fully automated PDB-wide analysis revealed many sequence
register errors in deposited cryo-EM models that would be otherwise very difficult to
spot owing to the size of the related models and limited resolution of the maps.
Finally, we show that findMySequence can assist manual model building into cryo-EM
maps at resolutions where poorly resolved side-chain moieties make both fully-
automated and visual map interpretation theoretically possible, but challenging using
available approaches. The method already proved crucial in building an atomic model
of the mycobacterial ESX-5 type VII secretion system into a 3.4 Å resolution cryo-EM
map [1].
15Carlos Oscar Sorzano
CSIC
Bias and variance in CryoEM
Cryo-Electron Microscopy (cryoEM) has become a well established technique to
elucidate the three-dimensional (3D) structure of biological macromolecules.
Projection images from thousands of macromolecules, assumed to be structurally
identical, are combined into a single 3D map that represents the Coulomb potential of
the macromolecule under study. In this article we discuss about possible caveats
along the image processing path and on how to avoid them in order to have a reliable
3D structure. Some of these problems are very well known in the community and we
may refer to them as sample related (like specimen denaturation at interfaces or non-
uniform projection geometry leading to underrepresented projection directions). The
rest are algorithmic related, and while some of them have been discussed in depth in
the literature, like using an incorrect choice of initial volume, there are others that have
received much less attention but, however, they are fundamental in any data analysis
approach. Chiefly among them we refer to instabilities in the estimation of many of the
key parameters required for a correct three-dimensional reconstruction that happen
all along the processing workflow and that may affect significantly the reliability of the
whole process.
In the field, the term overfitting has been coined to refer to some particular kind of
artifacts. We argue that overfitting is actually statistical bias in key steps of particle
estimation in the 3D reconstruction process, including intrinsic algorithmic bias. We
also show that common tools (FSC) and strategies (gold standard), that we normally
use to detect or prevent overfitting, do not fully protect us against it. Alternatively, we
propose that detecting the biases that lead to overfitting is much easier when
addressed at the level of parameter estimation, rather than detecting it once we have
combined the particle images into a 3D map. Parameter bias can be detected by
comparing the results from multiple algorithms (or at least, independent executions of
the same algorithm). Then, these multiple executions could be averaged in order to
have a lower variance estimate of the underlying parameters.
16Maya Topf
Birkbeck / CSSB Hamburg
Assessment and refinement of models from CASP14 cryo-EM
targets
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