IIMS Workshop - 3D_EM Data Format

Electron Microscopy Data Base (EMD): 3D-EM Macromolecular Structure Database

An Electron Microscopy Database has been set up at the European Bioinformatics Institute (EBI) under funding from the European Union project IIMS (http://www.ebi.ac.uk/pdbe/docs/IIMS.html)

What is the EMD aim?

EMD allows the management, organisation and dissemination of data on the structures of biological macromolecules solved by three-dimensional electron microscopy

The NEW 3D-EM database will provide a facility for storing volume maps, relevant textual descriptors and data files containing figures and sections. Where applicable the database will also contain layer-line data and structure factor files.

The database model has been integrated with the Macromolecular structure database at the EBI that also contains the PDB.

Will the data deposited in the EMD be accessible to anybody?

Yes. A release lock-in period can be placed on the 3D-EM map (up to 4 years) by the author, while the descriptive information will be released immediately (after it has been reviewed by the authors).

How can I deposit data in the EMD database?

The deposition system is now activated at:
http://www.ebi.ac.uk/pdbe-emdep/emdep

What is the information that can be deposited together with the 3D-EM map?

Apart from the 3D-EM map, other complementary information will be stored:

Textual descriptors:

Complementary data files:

A. Three orthogonal slices of the map (as image files, for easy
    visualization of the data)

    These will be mandatory and for immediate release. The author will
    choose the slices to be provided.

B. Supplementary figures (for illustrating important aspects)

C. 3D masks (for iso-surface rendering purposes; binary volume format)

    These will be optional and for immediate release.

D. Structure factors (only crystallography) and layer line data (only
    helical reconstruction)

What is the EMD map file format?

3D-EM volumes will be stored as binary files in CCP4 format.

During deposition, other EM map formats can be uploaded. These uploaded volume files will be automatically converted to CCP4 via the em2em program (http://www.imagescience.de/em2em/welcome.htm ). For very large volume files an ftp direct access can be allowed by notifying the EMD at: emdep@ebi.ac.uk.

Textual information

Together with the 3D-EM volume file, a set of textual annotations are included in the Electron Microscopy Database (EMD). This textual information is available for download in XML format. An EMD XML entry file is not only a well-formed XML document, but it also conforms to the EMD XML Schema.

For a complete definition of the EMD XML Schema, you can access:

The information relevant to an EMD entry will be contained in a element, and will be uniquely identified by its accession code. The general layout of an EMD entry file will be as follows:

<?xml version="1.0" enconding="UTF-8"?>

<emd_entry ...>
   <deposition> ... </deposition>
   <map> ... </map>
   <sample> ... </sample>
   <experiment> ... </experiment>
   <processing> ... </processing>
   <supplement> ... </supplement>
</emd_entry>

The contents of the main sections of information within an EMD entry, which have been included in the example above, are described in the following sections:

1. Deposition
2. Map
3. Sample
1. Sample component
1. Biochemical entity
4. Experiment
1. Sample preparation
2. Vitrification
3. Image recording
4. Image scanning
5. Processing
1. Reconstruction
2. Data set
6. Supplemental material

1. Deposition:

Contains context information relevant to the EMD entry record
XML tag <deposition>
XPath location: /emd_entry/deposition
Child elements:

<title>

Title: descriptive title for the EMD entry.

<entry_release_date>

Entry release date:
date of release of the EMD entry

<map_release_date>

Map release date:
date of release of 3D-EM volume file

<author_list>

List of authors:
list of authors of the deposition.
>> to be further described

<primary_reference>

Primary reference:
citation of the primary publication related to the 3D-EM
experiment
>> to be further described

<deposition>
   <title>3D volume of the DnaB helicase</title>
   <entry_release_date>2002-08-13</entry_release_date>
   <map_release_date>2002-08-13</map_release_date>
   <author_list num_auth="2">
      <author order="1">
         <last_name>San Martin</last_name>
         <name_initials>C.</name_initials>
      </author>
      <author order="2">
         <last_name>Carazo</last_name>
         <name_initials>J.M.</name_initials>
      </author>
   </author_list>
   <primary_reference pub_type="journal_article" published="1">
      <journal_article pmdid="9562559">
         <year>1998</year>
         <journal>Structure</journal>
         <volume>6</volume>
         <issue>4</issue>
         <first_page>501</first_page>
         <last_page>509</last_page>
         <article_title>Three-dimensional reconstructions from cryoelectron microscopy 
         images reveal an intimate complex between helicase DnaB and its loading partner
         DnaC
         </article_title>
         <authors>San Martin C, Radermacher M, Wolpensinger B, Engel A, Miles CS, 
         Dixon NE, Carazo JM
         </authors>
      </journal_article>
   </primary_reference>
</deposition>

2. Map

<file>

File:
name of the binary file where the 3D-EM volume is stored

Data type:
logical data type used for storing values of the 3D-EM volume (4-byte float, 2-byte integer, etc.).

Number of values (columns, rows and sections):
number of elements in the 3D-EM volume along each dimension.

Spacing [Å] (columns, rows and sections):
expressed in Angstroms, length of each value element in the 3D-EM volume along the three dimensions.

Origin [Å] (columns, rows and sections)
{not included for the moment}.

Data value statistics:
minimum, maximum, average and standard deviation values of the 3D-EM volume

Author's threshold:
threshold value proposed by the author to perform a surface rendering of the 3D-EM volume

Enforced symmetry:
symmetry constraints applied to the data values of the 3D-EM volume
>> to be further described

<map>
       <file class="map" format="ccp4">emd000100.map</file>
       <data_type>float</data_type>
       <num_row>50</num_row>
       <num_col>50</num_col>
       <num_sec>50</num_sec>
       <spacing_row units="A">3.8</spacing_row>
       <spacing_col units="A">3.8</spacing_col>
       <spacing_sec units="A">3.8</spacing_sec>
       <origin_row units="A">95.0</origin_row>
       <origin_col units="A">95.0</origin_col>
       <origin_sec units="A">95.0</origin_sec>
       <minimum>-9.1411295</minimum>
       <maximum>11.271612</maximum>
       <average>0.0001</average>
       <std>2.9369693</std>
       <auth_threshold>5.0</auth_threshold>
       <enforced_symmetry>
          <details>six-fold simmetry around Z axis</details>
       </enforced_symmetry>
</map>

3. Sample

Name:
descriptive name for the biological sample.

Aggregation state:
either single particle, icosahedral particle, helix, or 2D crystal.

Molecular weight [MDa]:
theoretical and experimental molecular weight of the sample. Details on the method used for experimental determination can be provided.

Details:
any other relevant information on the biological sample.

List of sample components:
A sample is further characterised by the detailed description of each of its distinct components (corresponding to child elements <sample_component>).

3.1. Sample component

Name:
descriptive name for the sample component (curated, using names found in relevant databases; see Nomenclature)

Author's name:
descriptive name for the sample component given by the author

Details:
any other relevant information on the sample component.

Nomenclature:
includes cross-references to relevant databases for nomenclature (GO for cellular components InterPro for proteins, and ICTVdb for viruses)
>> to be further described

Natural source:
for components directly isolated or purified from their source
>> to be further described

Compositional degree:

Virus:
in the case of a viral component, further information should be provided.
Child elements:

<empty>

Empty viral particle:
are the viral particles in the sample used to obtain the 3D-EM voluem empty? (i.e. they don't contain nucleic acid)

<enveloped>

Enveloped:
have the viral particles a lipid envelope?

3.1.1. Biochemical entity

Name:
descriptive name for the biochemical entity.

Oligomeric details:

Number of copies:

Details:
any other relevant information on the biochemical entity.

            3.1.1.a Molecular entity

<mol_entity> child elements:

<systematic_name>

 
Systematic name:

<common_name>

Common name:

<formula>

Formula:

            3.1.1.b Polypeptide entity

<poly_entity> child elements:
<mutant_flag>

Mutant flag:

<mutation_string>

Mutation string:

<fragment_flag>

Fragment flag:

<sequence>

Sequence:

<source>

Source:
A source is either a natural source or an engineered source:

Example:

<sample>
   <name>Dnab helicase hexamer</name>
   <aggregation_state>single particle</aggregation_state>
   <mol_wt_theo units="MDa">0.314340</mol_wt_theo>
   <sample_component_list num_comp="2">
      <sample_component id="ID000001" class="protein">
         <name>DnaB helicase</name>
         <auth_name>DnaB</auth_name>
         <details></details>
         <nomenclature class="InterPro (protein)">
            <ref_interpro _id="IPR001198">
               <name>DnaB helicase</name>
            </ref_go>
         </nomenclature>
         <comp_degree>exact</comp_degree>
         <entity_list num_elem="1">
            <entity id="ID000002" class="polypeptide entity">
               <name>String</name>
               <oligomeric_details>trimer of dimers</oligomeric_details>
               <number_copies>6</number_copies>
               <details>String</details>
               <poly_entity>
                   <sequence>
                   MAGNKPFNKQQAEPRERDPQVAGLKVPPHSIEAEQSVLGGLMLDNERWDDVAERVVADDF
                   YTRPHRHIFTEMARLQESGSPIDLITLAESLERQGQLDSVGGFAYLAELSKNTPSAANIS
                   AYADIVRERAVVREMISVANEIAEAGFDPQGRTSEDLLDLAESRVFKIAESRANKDEGPK
                   NIADVLDATVARIEQLFQQPHDGVTGVNTGYDDLNKKTAGLQPSDLIIVAARPSMGKTTF
                   AMNLVENAAMLQDKPVLIFSLEMPSEQIMMRSLASLSRVDQTKIRTGQLDDEDWARISGT
                   MGILLEKRNIYIDDSSGLTPTEVRSRARRIAREHGGIGLIMIDYLQLMRVPALSDNRTLE
                   IAEISRSLKALAKELNVPVVALSQLNRSLEQRADKRPVNSDLRESGSIEQDADLIMFIYR
                   DEVYHENSDLKGIAEIIIGKQRNGPIGTVRLTFNGQWSRFDNYAGPQYDDE
                   </sequence>
                   <source>
                      <engineered_source>
                         <organism>Escherichia Coli</organism>
                         <host_organism>Escherichia Coli</host_organism>
                         <host_vector>pPS560</host_vector>
                         <host_vector_type>plasmid</host_vector_type>
                   </source>
               </poly_entity>
            </entity>
          </entity_list>
       </sample_component>
    </sample_component_list>
</sample>

4. Experiment

4.1. Sample preparation

Buffer:

describes the composition of the sample buffer.
>> to be further described

Crystal growth:
describes the conditions for crystal growing.
>> to be further described

Sample support:
describes the support of the sample.
Child elements:
<film_material>

Film material

<method>

Method

<grid_material>

Grid material

<grid_mesh_size>

Grid mesh size

<grid_type>

Grid type

<pretreatment>

Pretreatment

<details>

Details

4.2. Vitrification

Cryogen:
substance used for rapid-freezing

Humidity [%]:
humidity (%) in the vicinity of the vitrification process

Temperature [Kelvin]

Instrument:
the type of instrument used in the vitrification process

Method:
description of the technique used for vitrification

Time resolved state:
The length of time after an event effecting the sample that vitrification was induced and a description of the event

Details:
any other relevant information on the vitrification process

4.3. Image recording

Microscope:
model and manufacturer of the electron microscope used.

Specimen holder:
details on the holder of the sample (type, model and/or manufacturer)

Accelerating voltage [kV]:
value of accelerating voltage (in kV) used for imaging

Illumination mode:
mode of illumination (e.g. flood beam, spot scan)

Imaging mode:
operating mode of the EM (e.g. bright field, dark field, diffraction)

Nominal Cs [mm]:
spherical aberration coefficient (Cs) in millimeters of the objective lens (nominal value)

Nominal defocus [ nm]:
defocus value of the objective lens (in nanometers) used to obtain the recorded images

Tilt angle [º]:
angle at which the specimen was tilted to obtain recorded images

Nominal magnification:
value of the magnification indicated by the microscope readout

Calibrated magnification:
magnification value obtained for a known standard just prior to, during or just after the imaging experiment

Electron source:
type of source of electrons (e.g. field emission, LaB6, tungsten)

Electron dose [electrons/ Å2]:
total electron dose received by the sample (electrons per square Angstrom)

Energy filter:
type of energy filter spectrometer apparatus

Energy window [eV]:
energy filter range in electron volts (eV) set by spectrometer.

Temperature [Kelvin]:
mean specimen stage temperature during imaging in the microscope

Detector:
detector used for recording images. Usually film or CCD camera.

Details:
any other relevant information on the image recording phase.

4.4. Image scanning

Scanner:
manufacture, model and/or type of instrument used for digitization.

Sampling size [microns]:
     sampling step size (microns) set on the scanner.

Details:
any other relevant information on the image scanning phase.

Example:

<experiment>
   <sample_preparation>
      <buffer>
         <details>
         protein samples were diluted (>50-fold) to 40 um/ml in 50 mM Tris.HCl pH 7.6,
         2mM DTT, 5mM MgCl2, 200 mM NaCl, 0.25 mM ADP
         </details>
      </buffer>
      <sample_support>
         <film_material>carbon</film_material>
         <grid_material>molybdenum</grid_material>
         <grid_type>holey</grid_type>
         <pretreatment>30 s of glow discharge</pretreatment>
      </sample_support>
   </sample_preparation>
   <vitrification>
      <gryogen_name>liquid ethane</gryogen_name>
      <instrument>double-side blotting device</instrument>
      <method>quick plunging</method>
   </vitrification>
   <imaging>
      <microscope>Philips EM 420</microscope>
      <accelerating_voltage units="kV">100.0</accelerating_voltage>
      <illumination_mode>spot scan</illumination_mode>
      <imaging_mode>bright-field</imaging_mode>
      <nominal_defocus_min units="nm">1.5</nominal_defocus_min>
      <nominal_defocus_max units="nm">1.5</nominal_defocus_max>
      <tilt_angle_min units="degrees">45.0</tilt_angle_min>
      <tilt_angle_max units="degrees">45.0</tilt_angle_max>
      <nominal_magnification>60000</nominal_magnification>
      <electron_dose units="e/A**2">10.0</electron_dose>
      <detector class="film">
         <detector_model>Kodak SO-163</detector_model>
      </detector>
   </imaging>
</experiment>

5. Processing:

5.1. Reconstruction

Method:
general method used for the 3d-reconstruction. E.g.: lattice line fitting, layer lines-Bessel functions, spherical harmonics, backprojection, ...

Algorithm:

Software:
information on the software packages used in the reconstruction procedure.
 >> to further described

CTF correction:
method used for Contrast Transfer Function (CTF) compensation

Resolution (by author) [Å]:
final resolution (in Angstroms)of the 3D reconstruction
Child elements: <resol_row> <resol_col> <resol_sec> <method>

Details:
any other relevant information on the reconstruction procedure.

5.2. Data set

5.2.a. 2D crystal:

Length (a, b, c) of unit cell [Å]:

Angles (alpha, beta, gamma) of unit cell [°]:

Two-sided plane group:

Details:

Structure factors:
>> to be further described

5.3.b. Icosahedron:

Number of digital images:

Number of projections:

T number:

Euler angle distribution:

Details:

5.2.c. Helix:

Delta phi:

Delta z:

Hand:

Axial symmetry:

Details:

Layer lines:
>> to be further described

5.2.d. Single particle:

<num_digital_images>

Number of digital images:

Number of projections:

T number:

Euler angle distribution:

Details:

Example:

<processing>
   <reconstruction>
      <method>angular refinement</method>
      <algorithm>algebraic reconstruction technique (ART)</algorithm>
      <software>
         <name>XMIPP</name>
      </software>
      <software>
         <name>SPIDER</name>
      </software>
      <resolution_by_author units="A">
         <resol_row>34.5</resol_row>
         <resol_col>34.5</resol_col>
         <resol_sec>34.5</resol_sec>
         <method>differential phase residual</method>
      </resolution_by_author>
   </reconstruction>
   <em_dataset>
      <single_particle>
         <num_digital_images>
         <num_projections>568</num_projections>
         <details>
         only a prevalent view of the sample could be detected 
         in the 0 degrees tilt micrographs
         </details>
      </single_particle>
   </em_dataset>
</processing>

6. Supplemental material (other data files)

Contains information on supplementary data items and material to the 3D-EM map
Corresponds to XML tag <supplement>
XPath location: /emd_entry/supplement
Child elements:

<slice_col> <slice_row> <slice_sec>

Slices:
three orthogonal slices of the map are provided for visualization purposes.

Masks:
a binary volume to segment or highlight relevant features of the 3D map (e.g.: specimen vs. background)

Figures:
any image with appropriate caption text to illustrate the 3D map.

Example:

<supplement>
   <slice_row num="25">
      <file class="image" format="CCP4">emd000100_row.map</file>
   </slice_row>
   <slice_col num="25">
      <file class="image" format="CCP4">emd000100_col.map</file>
   </slice_col>
   <slice_sec num="25">
      <file class="image" format="CCP4">emd000100_sec.map</file>
   </slice_sec>
   <figure_set num="2">
      <figure id="ID000003">
         <file class="image" format="gif">emd000100_f01.gif</file>
         <caption>
         Variation in the rotational energy corresponding to harmonics
         3 and 6 among successive slices of the reconstruction
         </caption>
      </figure>
      <figure id="ID000004">
         <file class="image" format="gif">emd000100_f02.gif</file>
         <caption>
         Tilt pairs of frozen hydrated DnaB. Untilted micrographs 
         on the left and 45° tilted micrographs on the right
         </caption>
      </figure>
   </figure_set>
   <mask_set num="1">
       <mask id="ID000005">
           <file class="mask" format="msk">emd000100_m01.msk</file>
           <caption>
           Mask corresponds to protein versus background, representing 100%
           of the expected volume (mean protein density: 1.33 g/cm3)
           </caption>
       </mask>
   </mask_set>
</supplement>